| Development the safe,non-toxic and stable performance of natural fungicide has become a research hotspot.Eucalyptus globulus as the introduction of fast-growing fiber plant in our country was widely cultivated in Sichuan,Yunnan and other provinces.Both in China and other growing areas E.globulus were the common folk herbal.Especially its rich E.globulus essential oils have good antibacterial,anti-inflammatory,analgesic and other biological characteristics.All of these characteristics were good materials for the development and utilization as new natural fungicides.The mechanism of essential oils from the E.globulus leaves has caused the attention of many scholarson bactericidal antibacterial,and it has become one of the hot microbiology researches in pathology,molecular biology,microbiology.The major postharvest disease of potato was dry rot,and it was also one of the main limitations of industrial development.Currently,the control of dry rot in the market was the use of chemical synthetic fungicides.However,in recent years due to the growing concern about food safety,and the constant social problems caused by a variety of chemicals,environmental issues occured,for example,water pollution,threaten life safety of human and animal.Due to the use of synthetic fungicides on a large scale,the resistance of strains to the chemical synthetic agents were increased,and eventually leading to the invalid of synthetic fungicides on strains,and its application was further limited.Therefore,researchers were actively looking for more appropriate and efficient safe fungicide,to gradually replace and reduce the application of hazardous chemicals fungicide.The experiment used Heilongjiang potato dry rot Fusarium as experimental materials,using organic chemistry,and molecular biology methods.The study of the E.globulus essential oils antimicrobial characteristics for the Fusarium,Purpose it has been applied in actual control of potato dry rot.The results were showed:1.The use of single-factor optimization test for E.globulus leaves oils extraction process.Pretreatment by enzyme,solvent-free vacuum extraction for E.globulus essential oils,the advantages of this process was that,after enzymatic pretreatment of raw material,the monoterpene glycosides of raw material were deglycosylated,thereby increasing the oil yield;at the same time,the enzyme treatment also causes the cell wall degradation and reduces the resistance of essential oils in releasing,accelerate distillation of essential oil.Not only the experiment was done under vacuum conditions,but also to ensure that the steam distillation was performed at lower temperatures to avoid the oxidation of essential oil components,and ultimately improve the overall extraction rate of E.globulus leaves oil.After enzyme pretreated E.globulus leaves of fresh,the raw material monoterpenes glycosides out of sugar,increase the oil yield;at the same time,enzyme processing the oil cell wall degradation,reducing the resistance to extract oil component,acceleration oils component distillation,essential oils were extracted by microwave under vacuum distillation to ensure that the distillation operation at a relatively low temperature,to avoid isomerization,oxidative denaturation oils components,and ultimately improve the overall E.globulus leaves oils extraction rateComparing before and after enzymatic treatment,the number and type of monosaccharide from E.globulus leaves in the ethanol extraction.we found that after the enzyme pretreatment,the monosaccharide types has not changed,but the number of monosaccharide has increased significantly,almost 2 times the original;at the same time,comparing the different extraction methods of E.globulus leaves by scanning electron microscope,epidermal cells of leaves have changed to varing degrees,the arrangement of stomatal,surface cells and the surface wax were different.The mositure of E.globulus leaves,extraction time,microwave power were studied and a total of three factors were chosen to establish a good fit model and equation.According to the equation,the optimum extraction conditions of essential oils were shown:moisture contenr 60%,extraction time 27 min,microwave power 548 W.Under these conditions the yield of oil was 6.98 mL/kg,the precision of this model was 21.78,the relative standard deviation of this method 1.68%.After the evaluation system,this innovation technology of E.globulus leaves oils extraction not only enhance the yield of essential oils,but also their properties well protected,it indicated that the extraction process was reasonable and reliable,for the separation of essential oil components,there was provided a new idea of green.Enzyme pretreatment-solvent-free vacuum microwave extraction of essential oils,has been reported by the less literature search.2.Using E.globulus leaves oils inhibit the potato dry rot pathogen Fusarium experiments,we found that E.globulus leaves oils and its main monomer components can suppressed mycelial growth,spore production,germination and development role.The minimum inhibitory concentration(MIC)were:F.trichothecioides 2 ?L/mL,F.sporotrioides 1.25 ?L/mL,F.culmorum 0.75 ?L/mL,F.solani var coeruleum 2 ?L/mL,F.avenaceum 1.5 ?L/mL.The most sensitive pathogens for the essential oils of E.globulus were:F.culmorum,F.sporotrioides.Their minimum bactericidal concentration(MBC)were:0.75 ?L/mL,1.5 ?L/mL,respectively.Eucalyptus essential oils were used to inhibit the growth of Fusarium,the same study reported has less literature.The inhibitory effect of major monomer component on sensitive strains were different:a-terpineol and terpinyl acetate for F.sporotrioides has a strong inhibitory effect,a-terpineol and eucalyptol for F.culmorum has obvious inhibition.Experiments showed that E.globulus leaves oils and its main compounds for dry rot bacteria have a common effect,which can effectively inhibit the Fusarium growth,reproduction,and also showed that the main part of the essential oils content were not entirely positive association,not its content,the stronger antibacterial ability not means the higher components,and the other oxygen-containing compounds of the single terpenoids were highly correlated,the mutual concurrent effects on antibacterial ablility.Taken together,a-terpineol have good inhibitory effect for the several Fusarium,a-terpineol used to suppress Fusarium,less been reported by the literature search,the fact provide reference for other rich a-terpineol essential oils of plants.3.Different concentrations of E.globulus leaves oils and inoculated in the medium and Fusarium spore suspension,which can effectively control the growth of fungi of hyphae and spores,which E.globulus leaves oils intended for F.sporotrioides and F.culmorum the best effect.In vitro tests,we found that the essential oils of E.globulus leaves can inhibit dry rot fungi colony growth,mycelial growth and full germination.By scanning electron microscopy showed that E.globulus leaves oils processing mycelium can induce morphological changes,manifested as hyphae twisted,wound,swollen hyphae exhibit wilting,weak,wrinkled,irregular thickening or breakage,deformation and other anomalies morphological changes.Through an electron microscope observed,after E.globulus leaves essential oils dispose the Fusarium,a lot of spore germination were inhibited,it showed short spore germ tubes or deformity,spore forming transparent vacuoles.With increasing concentration of essential oils,the extent of mycelial cell damage will be greater,the disintegration of the mycelium will be more serious,leading to leakage of protoplasts,conductivity and MDA of the values were elevated,necrosis hyphae and spores increase in the number,indicating that E.globulus leaves oils processing enables the mycelium morphological changes,structural damage,and finally to the death of bacteria.4.E.globulus essential oils and its components research on the machanism of Fusarium DNA.E.globulus leaves oisl and its main monomer compounds for dry rot pathogen inhibition have different at the molecular level.By gel electrophoresis and UV spectrophotometer to detect the expression changes in their DNA,The experiment results showed that;After essential oils and main monomer compound treat the F.sporotrioides and F.culmorum had fallen to total DNA content and concentration,control group proposed F.sporotrioides the amount of DNA was 1.29 ?g,the eucalyptol,terpineol,terpinyl acetate after treatment respectively,the total DNA:1.09,1.00,and 1.06 ?g,control group F.culmorum,the amount of DNA was 1.22 ?g was significantly higher than other essential oils and the DNA content of monomer after processing hyphae,gel electrophoresis test results also showed that E.globulus leaves oils,?-terpineol,terpinyl acetate,borneol,eucalyptol,?-pinene types were weak the control group as clear and bright.It showed that the dry rot pathogen DNA synthesis,replication,and proteins of guidance damaged by essential oils,making it impossible complete the normal metabolism of cells,so that the proposed F.sporotrioides and F.culmorum was suppressed.In summary,the method of enzyme pretreatment-without solvent vacuum extract E.globulus essential oils,it not only ensured essential oils improve the overall yield,but aslo the monomers composition characteristics were keeped,after deal with the dry rot pathogens,Experiments showed that E.globulus leaves essential oils and its main monomer compound can inhibit the potato dry rot pathogen Fusarium growth and reproduction,reduced spore germination rate,and reduce damaging effects of the potato.In the study of essential oils damage cell membrane into the Fusarium cell,by scanning electron microscopy(SEM)and optical microscopy observed morphogenesis distortion of hyphae and spores,it showed that essential oil damage Fusarium at the molecular level,and by gel electrophoresis observed that E.globulus essential oil maked Fusarium decreased expression of DNA synthesis,and also on the level of DNA damage. |
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