| Agriculture is the source of human’s basic living materials, and is the critical domain of the development of national economy. China’s grain production has increased for successive 12 years under the protection of pesticide. However, the problem of environmental pollution caused by pesticides have become increasingly prominent as we got the gratifying achievements, which influences severely on human health. Plant secondary metabolites are evolved during the process of adapting to the environment and resisting the invasion of pests and diseases, it is an important resource for screening green pesticides because it has a series unique advantage such as abundant in natural resource, renewable and good compatibility with the environment.In this paper, we described the studies on antimicrobial activity and mechanism against plant pathogens of sesquiterpenoids from E. adenophorum, we also explored the distribution, accumulation dynamics and hydrolysis of two sesquiterpenoids. The main results are as following:1、Three steps to obtain E. adenophorum leaf oil, first extracted with methanol, followed by extracted with ethyl acetate, finally purified using silica gel column. Gas chromatography mass spectrometry(GC-MS) was used to separate and identify the components of leaf oil, and the relative content of the components were determinated by area normalization. GC-MS analysis resulted in the identification of twelve compounds representing 99.15% of the total oil composition. The main components were oxygenated sesquiterpenes accounted for 98.17%, including 10Hβ-9-oxo-agerophorone(37.03%), 10Hα-9-oxo-agerophorone(37.73%) and 9-oxo-10, 11-dehydro-agerophorone(23.41%).2、A poisoned food technique was used to evaluate antifungal activity of E. adenophorum leaf oil against four tested pathogenic fungi. Our results showed that E. adenophorum leaves oil was particularly active against Pythium myriotylum and Phytophthora capsici, with minimal inhibitory concentration of 0.1mg/m L and 0.5mg/m L respectively, while A. tenuissima and F. solani appeared less sensitive to the oil. Moreover, the effect of E. adenophorum leaves oil was examined under a light microscope to study the mode of action on P. myriotylum, after exposure to leaves oil, the hypha diameter increased, cell wall decay, cytoplasmic contents were distributed heterogeneously, cytoplasm granulation and cytoplasm decrease were clearly visible. The effect of E. adenophorum leaves oil on inhibition of decay development in artificially inoculated infected ginger was tested in vivo, which showed leaf oil can control ginger decay at 0.2mg/m L concentrations.3、There was a certain synergism of E. adenophorum leaves oil mixed with mefenoxam, mancozeb and iprodione separately in controlling mycelium growth of P. myriotylum and P. capsici.4、In order to investigate the mechanism of E. adenophorum leaf oil against oomycete, we examined its effect on MDA, soluble sugar, soluble protein, SOD, CAT, POD, SDH and MDH in P. myriotylum. Moreover, the influence of E. adenophorum leaves oil on the ultrastructure of P. capsici and P. myriotylum were observed by transmission electron microscopy, and on cell membrane permeability of P. capsici was measured by electrical conductivity method. The results indicated that the activities of SOD, CAT, POD and SDH of P. myriotylum were reduced and the activities of MDH was increased by E. adenophorum leaf oil, it means that E. adenophorum leaf oil against P. myriotylum by reducing the cell’s ability to clear free radicals and destroying the three carboxylic acid cycle. The content of MDA increased indicated that leaf oil induced the reaction of lipid per oxidation on the membrane of P. myriotylum and damaged cell membranes. At the concentrations of 0.06mg/m L and 0.08mg/m L, leaf oil decreased the content of soluble sugar, soluble protein increased first and decreased afterward in cell of P. myriotylum, which reflected the leaf oil affected biosynthesis in the cell. Cells treated with leaf oil exhibited typical markers of apoptosis, such as cell rupture, degradation of cell organelle, cell contents decrease, empty cavity cell. The relative conductance in treatment group was higher than control group indicated that E. adenophorum leaves oil changed the cell membrane permeability of P.capsici.5、Two cadinene sesquiterpene named 10Hβ-9-oxo-ageraphorone and 9-oxo-10,11-dehydroageraphorone were isolated from the leaves of E. adenophorum. All of them showed good antifungal efficacy against common plant pathogens, including Alternaria tenuissima, Fusarium oxysporum, Fusarium proliferatum, Bipolaris sorokiniana and P. myriotylum. They are also effective in inhibiting spore germination of F. oxysporum. Our scanning electron microscopy study revealed 10Hβ-9-oxo-ageraphorone and 9-oxo-10, 11- dehydroageraphorone against pathogens by destroying the structure of hyphae and accelerating aging of hyphae and spores. However, the antifungal activity of 10Hβ-9-oxo-ageraphorone and 9-oxo-10,11-dehydroageraphorone was obviously less than their mixture—E. adenophorum leaf oil.6、Determination of minimal inhibitory concentrations demonstrated that 10Hβ-9-oxo-ageraphorone and 9-oxo-10,11-dehydroageraphorone exhibited low bactericidal activity against race 4 of Ralstonia solanacearum, but 9-oxo-10,11-dehydroageraphorone exhibited well bactericidal activity against race 1(R4) with MIC values of 0.25mg/m L.7、UPLC-MS/MS method was established to analyze the distribution and dynamic accumulation of 10Hβ-9-oxo-ageraphorone and 9-oxo-10,11-dehydroageraphorone in E. adenophorum. Their hydrolysis rule were also determined. The results indicated that the two sesquiterpene are mainly distributed in leaves of E. adenophorum, and the content of the two sesquiterpene were higher in vegetative period, less in reproductive period. The two sesquiterpene were easy to be hydrolyzed and the hydrolysis rate was less affected by temperature. |
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