Inhibition Mechanism Of Palmarosa Essential Oil Against Gray Mold Disease Of Onion And Construction Of Nano-Sustained Release System

The onions are perennial herbaceous plants which belongs to the lily family,Amaryllidaceae,and the genus,Allium.It is rich in nutrients and high moisture content,and is susceptible to fungal infestation during storage.Among them,gray mold caused by Botrytis cinerea(B.cinerea)is a serious fungal disease in storage.Natural plant essential oils have broad-spectrum antifungal properties and can inhibit a variety of pathogenic fungi.Palmarosa essential oil(PEO),as a common plant essential oil,has strong antifungal activity,but there are few reports on the control of gray mold of onion caused by B.cinerea.Therefore,in this experiment,B.cinerea was used as the test strain and onion as the test material.In vitro the antifungal activities of plant essential oils against B.cinerea were investigated,changes in mycelial morphology were observed before and after the treatment,the control effect of PEO on onion gray mold was evaluated by onion bulb test.The effect of PEO on the cell membrane integrity and permeability of the fungus was analyzed,and the changes of malondialdehyde content,reactive oxygen species content and antioxidant enzymes activity in the fungus were further examined to investigate the mechanism of inhibition.Finally,mesoporous nanosilica(MCM41)was synthesized,and the slow release of PEO was achieved by modifying mesoporous silica for later practical application.The main results of the study are as follows:(1)Screening of antifungal essential oils.B.cinerea was used as the test strain,and the inhibition ability of PEO,turpentine oil and lemon oil were investigated.The results showed that all three oils could effectively inhibit the growth of B.cinerea colonies.0.50 mL L-1 of PEO completely inhibited the mycelial growth of B.cinerea,while turpentine and lemon oil inhibited the mycelial growth of B.cinerea at 12.8 mL L-1 and 6.4 mL L-1,respectively.The MIC values of the three essential oils were PEO(0.30 mL L-1)<lemon oil(6.40 mL L-1)<turpentine oil(12.8 mL L-1).The PEO was chosen for later experiment due to its strong inhibitory activity.The inhibition rate of B.cinerea mycelium was 29.48%,52.12%and 100%at the concentration of 0.30 mL L-1,0.40 mL L-1 and 0.50 mL L-1 of PEO,respectively,and the EC50 value of PEO was calculated to be 0.31 mL L-1 based on the mycelial inhibition test.The EC50 value of PEO was used to reset the subsequent test gradients,and the germination inhibition rates of B.cinerea were 38.10%,60.0%,92.86%and 100%at different concentrations of PEO(0.15 mL L-1,0.30 mL L-1,0.45 mL L-1 and 0.60 mL L-1)in the spore germination inhibition test,respectively.GC/MS analysis of PEO showed that the main active ingredient was citronellol(44.67%),and other substances with a higher percentage of components were benzyl benzoate(14.66%),acetyl cedrene(9.63%),geranylgeraniol(8.58%),and geraniol(6.82%).(2)The inhibition mechanism of B.cinerea was investigated with PEO.SEM observation showed that the mycelium of B.cinerea showed different degrees of deformation,wrinkling and even collapse after PEO treatment.The results of extracellular conductivity,cell contents and ergosterol synthesis showed that the treatment of PEO against B.cinerea disrupted the integrity of cell membrane and changed the permeability of cell membrane,resulting in the increase of extracellular conductivity and the leakage of nucleic acid and protein.The ROS staining,MDA content and antioxidant enzyme POD assay showed that a large amount of reactive oxygen species was produced in the fungi after PEO treatment,and the MDA content increased significantly and the POD content decreased significantly.It is assumed that the oxidative stress was generated in B.cinerea after treatment with PEO,resulting in a large accumulation of peroxidation products(MDA).In addition,the reduced activity of POD led to the failure of peroxidation products to be decomposed in time,which further aggravated the membrane lipid peroxidation of B.cinerea and eventually led to the abnormal growth or direct death of B.cinerea.The efficacy test of onion gray mold showed that PEO treatment could effectively reduce the size of the spot diameter of onion gray mold.The inhibition rate of gray mold in the treatment group reached 51.27%,68.54%,82.91%and 99.07%at different concentrations of PEO treatment onion(0.15 mL L-1,0.30 mL L-1,0.45 mL L-1 and 0.60 mL L1).In the prevention group,the inhibition rates were 63.86%,72.78%,84.03%and 100%,respectively.The prevention group was found to be more effective than the treatment group.In addition,PEO also enhanced the defense of onion against gray mold by inducing an increase in peroxidase activity in onion bulbs.(3)MCM41 was synthesized and modified by VTMO in room temperature,and the particle size,zeta potential and nitrogen adsorption desorption analysis revealed that the particle size of VTMO modified MCM41 became larger,the specific surface area decreased and the potential decreased,which further confirmed the successful modification of VTMO.The loading performance analysis revealed that the loading ratio of MCM41,VTMO-MCM41 on PEO were 72.65%and 29.52%,respectively.The slow-release performance test revealed that the cumulative release rate of PEO and PEO-MCM41 reached 50%at 16 h and 20 h,respectively,while the cumulative release rate of PEO-VTMO-MCM41 was only 39.22%after 24 h.The above results indicate that the VTMO-modified mesoporous silica significantly improved the slow-release performance of PEO.Compared with the control group and PEO.the inhibition test revealed that the inhibition time of B.cinerea by PEO-MCM41 and PEOVTMO-MCM41 increased significantly by 7 d and 14 d,respectively.All these results indicated that the VTMO-modified mesoporous silica significantly improved the slow-release performance of PEO and initially achieved its long-lasting inhibition performance.