The Control Effect And Mechanism Of Essential Oils From Several Chinese Traditional Medicinal Crops On The Disease Of Panax Notoginseng

Background Panax notoginseng(Burk.)F.H.Chen,belongs to the Panax genus of Araliaceae family,is the most characteristic biological resource in Yunnan Province.The plant has the functions of activating blood circulation,alleviating pain and mainly distributed in Wenshan.In recent years,the planting area increases sharply which leads to more and more serious diseases,and continuous cropping obstacle has become a bottleneck problem hindering the healthy development of Chinese traditional medicine.In addition to root-rot diseases,it's vulnerable to the above-ground diseases due to its particular growth environment,such as grey mould diseases and round-spot diseases and so on,and those diseases become a vital factors limiting the yield and quality of P.notoginseng.At present,the control of P.notoginseng fungal infection frequently relies on the chemical methods.Although the chemical methods can achieve effective control effect in a short time,it can cause "3R" problems easily,which is not in line with the modern concept of green agricultural development.Therefore,in order to avoid pesticide residues,environmental pollution and other issues,it has become a research hotspot to extract natural antibacterial products from plants.In view of the adverse effects of chemical fungicides,so hope to find a green and effective plant-based pesticide to replace the application of chemical fungicides.And the action mechanism needs more experiments to be done to lay a solid theoretical foundation for the development of new green pesticides or chemical fertilizers.Object In this paper,the essential oils of some representative Chinese medicinal crops of Rutaceae,Umbelliferae and Labiatae were taken as research objects,and the common pathogens of P.notoginseng diseases were taken as the representative strains to study the inhibition of essential oils from Chinese medicinal crops on the growth of P.notoginseng diseases pathogens,which could clarify the action mechanism of the inhibition of essential oils from Chinese medicinal crops on P.notoginseng fungi.So that solving the problems of continuous cropping obstacles,realizing standardized planting,improving the output and quality of P.notoginseng,and increasing the economic benefits of growers.Method1.The morphological characters of mycelia and spores of seven pathogens of P.notoginseng diseases were observed by microscope.2.The five essential oils from Rutaceae,Cumin,Mentha haplocalyx and Schizonepeta tenuifolia were extracted by steam distillation,and the antifungal effects in vitro on P.notoginseng fungi were carried out by the Oxford cup method.3.The main chemical compositions and contents of Citrus medica,Cumin,M.haplocalyx and S.tenuifolia essential oils were analyzed by GC-MS.4.The minimum inhibitory concentration of C.medica,Cumin,M.haplocalyx and S.tenuifolia essential oils and their main chemical compositions were determined by 96-well plate method.5.The synergistic effect of the essential oils of Chinese traditional medicinal crops(C.medica,Cumin,M.haplocalyx and S.tenuifolia)and hymexazol on the pathogenic fungi of P.notoginseng was determined by chessboard method.6.The growth effects of essential oils from C.medica,Cumin and M.haplocalyx on the P.notoginseng seedlings in vivo,which inoculated with Fusarium oxysporum,were determined by pot method.7.The F.oxysporum were treated with Cumin and M.haplocalyx essential oils,then the internal structure and morphology of mycelia and spores were observed by scanning electron microscope(SEM)and transmission electron microscope(TEM).8.The effects of spore germination rates,mycelia dry weights,spore yields,mycelia soluble protein contents,reducing sugar contents,relative electrical conductivity,SDH and NADH oxidase of F.oxysporum treated with different concentrations of M.haplocalyx essential oil were determined.Results1.The colony morphology,mycelia structure and spore morphology of F.oxysporum,Fusarium solani,Cylindrocarpon destructans,Pythium aphanidermatum,Botrytis cinerea,Colletotrichum gloeosporioides and Rhizoctonia solani were consistent with that of the previous literature's.2.The essential oils of five kinds of Chinese medicinal crops from Rutaceae were extracted and carried out antifungal activities test.The results showed that C.medica essential oil was found to have stronger inhibitory effects on the growth of pathogenic fungi in vitro than other four essential oils;The essential oils of Cumin,M.haplocalyx and S.tenuifolia were extracted.The results of Oxford cup methods showed that Cumin,M.haplocalyx and S.tenuifolia essential oils had strong antifungal effect on seven P.notoginseng fungi at the concentration of 50 mg/m L,and the inhibitory rates were 100 %.3.The main chemical constituents of C.medica,Cumin,M.haplocalyx and S.tenuifolia essential oils were analyzed.The results showed that the five predominant constituents of C.medica essential oil were d-limonene(22.79 %),?-terpinene(9.71 %),geraniol(5.77 %),nerol(5.54 %)and(R)-(+)-?-terpineol(5.10 %);The main constituents of Cumin essential oil were cuminaldehyde(44.53 %),p-cymene(12.14 %),p-mentha-1,3-dien-7-al(10.63 %),(-)-?-pinene(10.47 %)and ?-terpinene(8.40 %);The main compositions of dry M.haplocalyx essential oil were D-carvone(29.71%)and d-limonene(7.86%),and the main compositions of fresh M.haplocalyx were D-carvone(37.87 %)and d-limonene(9.98 %);The main compounds of dry S.tenuifolia essential oil were(-)-menthone(39.23 %),p-menthan-3-one(30.28 %)and d-limonene(6.95 %).4.The MIC assay results showed that essential oils of C.medica,Cumin,M. haplocalyx and S.tenuifolia and their major compounds had strong inhibitory effects against the P.notoginseng fungi,and had selective inhibition effect on different fungi.Among them,the inhibitory effect of cuminaldehyde from Cumin had significant antifungal activities,which reached the same order of magnitude as the hymexazol.5.It's a reasonable way in analyze the synergistic effect between essential oils and hymexazol to increase their antimicrobial activity by chessboard method.According to the FIC index values,the results have indicated that the C.medica essential oil and hymexazol showed no effect against F.oxysporum,a synergistic effect on F.solani,and an additive effect on C.destructans;And combined use of Cumin essential oil and hymexazol shown synergistic inhibitory effect on P.aphanidermatum,while the effects on F.solani were additive,and the result showed irrelevant effect on inhibiting B.cinerea,C.destructans,F.oxysporum,C.gloeosporioides;A synergistic effect between M.haplocalyx essential oil and hymexazol were found against P.aphanidermatum,and no relevant effect was perceived against the other fungi;The combination of S.tenuifolia essential oil and hymexazol showed a synergistic effect on P.aphanidermatum,and the effect against F.solani was additive.There were no relevant effects on F.oxysporum,C.destructans,B.cinerea,or C.gloeosporioides.6.The results of in vivo experiments showed that added essential oils(C.medica,Cumin and M.haplocalyx)could significantly reduce the disease incidence and index of P.notoginseng,and the growth of P.notoginseng were relatively better,which were no significant difference compare with control treatment.7.In SEM treatment,the control treatment shown that the spore and mycelia of F.oxysporum were normal with a smooth and complete shape,while the number of F.oxysporum spores were decreased sharply and obvious detrimental effects on the morphology of mycelia and spores were shown when F.oxysporum were treated with Cumin essential oil,cuminaldehyde and M.haplocalyx essential oil;The SEM results showed that the spores morphology in control group had the typical structure,such as,cell membrane,cell wall and internal organelles were visible clearly.Added Cumin essential oil,cuminaldehyde and M.haplocalyx essential oil,cells were shriveled and crumpled,internal organelles were dissolved,and plasmolysis became more and more serious.8.F.oxysporum was chosen as dominant fungi to investigate the action mechanism of M.haplocalyx essential oil.The results shown the different concentrations of M.haplocalyx essential oil(2 MIC,MIC,1/2 MIC,1/4 MIC,1/8 MIC and 1/16 MIC)inhibited the spore germination rates,mycelia dry weights,spore yields;And the mycelia soluble protein contents,reducing sugar contents and relative conductivity were increased because destroyed the cell structure;What's worse,the M.haplocalyx essential oil suppressed the activity of SDH and NADH oxidase,hindering the respiratory chain in F.oxysporum.Conclusion This study demonstrated that C.medica,Cumin,M.haplocalyx and S.tenuifolia essential oils and their main compounds had excellent antifungal activities against the pathogenic fungi of P.notoginseng in vivo and in vitro.There were good synergistic effects between the essential oils and hymexazol against some P.notoginseng pathogens.Thus the essential oils could decrease the incidence and index diseases of P.notoginseng.In the present study,we were selected F.oxysporum as the representative strain and explored the action mechanism of M.haplocalyx essential oil through the following experiments.The spore germination rates,mycelia dry weights and spore yields sharply decreased with increasing M.haplocalyx essential oil concentrations.The mycelia soluble protein contents,reducing sugar contents and relative conductivity of F.oxysporum gradually increased,because the M.haplocalyx essential oil destroyed the integrity and permeability of the cell membrane system,and verified that the membrane system may be one of the main targets of plant essential oils;In addition,the activities of the enzymes related to the respiratory chain of F.oxysporum(SDH and NADH)decreased,indicating that inhibiting the respiration of F.oxysporum may be one of the antifungal mechanisms of M.haplocalyx essential oil.