Fungicidal Activity And Mechanism Of Three Plant Essential Oils Against Fusarium Solani Associated With Astragalus Membranaceus Root Rot

Astragalus membranaceus is a leguminous perennial herb with extreme economic and medicinal value.In recent years,A.membranaceus root rot has been seriously occurred in weiyuan,longxi of Gansu province and hunyuan of Shanxi province,causing huge losses in yield and quality.At present,A.membranaceus root rot is controlled by crop rotation and chemical fungicides,but the control effect is very low.Therefore,this study aimed to find and develop an effective broad-spectrum bio-fungicide to control A.membranaceus root rot.In vitro the antifungal activities of plant essential oils against Fusarium solani were determined.Observing the morphological changes of F.solani,the effects of essential oils on the integrity and permeability of cell membranes were analyzed.Further,the changes of malondialdehyde content,mitochondrial membrane potential,reactive oxygen species content and the antioxidant enzyme activity in fungi were explored.Finally,the control effect of essential oils on A.membranaceus root rot was tested through pot experiment.The main findings were as follows:（1）The results of GC-MS exhibited that cinnamaldehyde（C₉H₈O）was the main compound of cinnamon essential oil with a relative content of 82.90%,and eugenol(C₁₀H₁₂O₂)in clove essential oil as the main compound with a relative content of 70.63%,the main component of oregano essential oil was carvacrol(C₁₀H₁₄O,77.65%).Six essential oils had inhibitory effects on F.solani by the minimum inhibitory concentration method,and the order of antifungal activity was from strong to weak:carvacrol,oregano essential oil(200μL L^-1)>cinnamaldehyde(300μL L^-1)>cinnamon essential oil(350μL L^-1)>Eugenol(400μL L^-1)>clove essential oil(500μL L^-1).（2）The results in vitro showed that cinnamaldehyde,carvacrol and eugenol could effectively inhibit the mycelial growth of F.solani in a dose-dependent manner,with EC₅₀ of178.68,92.39 and 263.00μL L^-1,respectively.In addition,three essential oils could inhibit the spore germination of F.solani.When the concentrations of cinnamaldehyde,carvacrol and eugenol were 120 160 and 140μL L^-1,respectively,the inhibition rates of spore germination were 99.61%,98.42%and 94.18%,respectively.The mycelia of F.solani treated with essential oils showed irregular shrinkage along with obvious wizened,depression and fracture by SEM.Propidium iodide staining showed that the cell membrane integrity of F.solani was impaired treated with essential oil.Meanwhile,the electrical conductivity and protein in the supernatant were positive with the treatment time and the concentration of essential oils,indicating that the permeability of cell membrane of F.solani was damaged.Three essential oils prominently inhibited the synthesis of ergosterol in F.solani cell,and the reduction rate of ergosterol content was positively correlated with the concentration of essential oils.（3）Cinnamaldehyde and eugenol could cause oxidative stress in F.solani,resulting in a significant increment of MDA content,a decrease in mitochondrial membrane potential and a large accumulation of reactive oxygen species.RT-q PCR results showed that cinnamaldehyde could significantly up-regulate the expression level of Fs Nox R,and eugenol could up-regulate the expression levels of Fs Nox R,Fs Rac and Fs Nox B so that numerous reactive oxygen species were produced by NADPH oxidase.In addition,cinnamaldehyde and eugenol inhibited the activities of POD and SOD,compared with the control group,600μL L^-1 cinnamaldehyde inhibited the activities of POD and SOD in F.solani by 28.69%and 41.62%,respectively,and the inhibition rates of 800μL L^-1 eugenol on POD and SOD were 33.10%and 34.15%,respectively.（4）Cinnamaldehyde and eugenol significantly reduced the activities of F.solani cellulase and pectinase so that the pathogenicity of F.solani was reduced.In vivo experiments proved that the treatments of essential oils significantly reduced the infection and colonization of F.solani in A.membranaceus roots,and reduced the lesion area in concentration-dependent.After treatment with 600μL L^-1 cinnamaldehyde and 800μL L^-1 eugenol for 7 days,the lesion areas of root rot were reduced by 93.97%and 89.64%,respectively,compared with control groups.The pot experiment showed that the control effects of the treatment group and the prevention group treated with 600μL L^-1 cinnamaldehyde were 92.98%and 83.33%,respectively.Similarly,that of 800μL L^-1 eugenol was 85.45%and 84.55%,respectively.Thus,cinnamaldehyde and eugenol can be used for the control of A.membranaceus root rot.