| BackgroundThe root rot of Panax notoginseng(P.notoginseng)caused by the infection of pathogenic fungi has seriously affected the quality and yield of P.notoginseng.At present,chemical pesticide was mainly used to control this disease.But the chemical drugs are easy to cause harm to the environment and human body,so to find green natural bacteriostatic agents has always been the primary task of the development of P.notoginseng planting industry.Due to its low residue,non-pollution and perfect extraction method,plant essential oils(EOs)was gradually used in the field of agricultural fields,and was considered as a candidate for the development of new pesticides.Plant EOs can inhibit fungal growth and reduce fungal pathogenicity in a variety of ways.Basic research on the antifungal mechanism of plant EOs has mainly focused on fungal cell structure.However,the molecular mechanism of how plant essential oils target fungi is relatively unknown.ObjectMentha haplocalyx(M.haplocalyx)EO was taken as the research object in this paper,and F.proliferatum isolated for the first time from the root rot of P.notoginseng was taken as the representative strain that cause the disease.The inhibitory effect of M.haplocalyx EO on F.proliferatum was researched,and further study the molecular mechanism of M.haplocalyx EO inhibiting F.proliferatum,which provide the experimental basis for the development of green pesticides/fertilizers,and provide the theoretical basis for the prevention and control of the root rot of P.notoginseng.Methods1.The EO from M.haplocalyx was extracted by steam distillation.The minimum inhibitory concentration(MIC)of the M.haplocalyx EO against F.proliferatum was determined by 96-well plate method.2.The inhibitory effect of M.haplocalyx EO on spore germination rate,spore vitality,spore yield and dry weight of mycelium of F.proliferatum was determined.3.The effect of M.haplocalyx EO on the membrane permeability and the activity of cell wall degrading enzymes of F.proliferatum were determined.4.The effect of M.haplocalyx EO on the gene transcription level of F.proliferatum was determined by transcriptomics.5.The effect of M.haplocalyx EO on histone modification was determined by Western Blot.6.The effect of M.haplocalyx EO on the acetylation level of histone protein of F.proliferatum was determined by using ChIP coupling technology.7.Combined analysis of transcriptomics and ChIP-seq to find the target of action of M.haplocalyx EO inhibiting F.proliferatum.8.The effect of M.haplocalyx EO and main ingredient carvone on histone deacetylase gene expression.Results1.The fungus was F.proliferatum OP430570.The MIC of M.haplocalyx EO to F.proliferatum was 0.59 mg/m L.2.M.haplocalyx EO can reduce the spore germination and spore vitality of F.proliferatum,inhibit the increase of spore yield and dry weight of mycelium,and the spore germination rate,spore vitality,spore yield and dry weight of mycelium of F.proliferatum decrease with the increase of M.haplocalyx EO concentration.3.M.haplocalyx EO can destroy the cell membrane of F.proliferatum increase the permeability of the cell membrane,and increase the content of reducing sugar,soluble protein and relative conductivity in the solution.M.haplocalyx EO can reduce the activity of cell wall degrading enzyme of F.proliferatum,thus reducing the pathogenicity of F.proliferatum.The higher the concentration of M.haplocalyx EO,the lower the activity of cell wall degrading enzyme.4.M.haplocalyx EO has a great influence on the transcription level of F.proliferatum genes.Compared with the control group,5471 differentially expressed genes were detected in MIC treatment group,including 2755 up-regulated genes and 2716 down-regulated genes;A total of 2501 differentially expressed genes were detected in the 1/8 MIC treatment group,and the up-regulated/down-regulated genes were 1251/1250 respectively.Through KEGG enrichment analysis,it was found that the enrichment of down-regulated differentially expressed genes was concentrated in the ribosomal pathway,the enrichment of up-regulated differentially expressed genes was concentrated in the DNA and protein repair pathway.5.M.haplocalyx EO inhibits the acetylation modification level of histone H3 of F.proliferatum.6.ChIP-seq data showed that the difference peak of acetylation modification was mainly distributed in the TSS and promoter region.Compared with the control group,H3K27 ac of MIC treatment group had a total of 254 up-regulated peak numbers and 503down-regulated peak numbers.H3K9 ac of MIC treatment group had 261 up-regulated peak numbers and 388 down-regulated peak numbers.In general,after the treatment of peppermint essential oil with MIC concentration,The acetylation level has a downward trend.KEGG enrichment of down-regulated genes revealed that down-regulated genes were mainly concentrated in ribosomal pathway.7.The down-regulated differentially expressed genes and differentially modified genes were intersected.There were 86 co-down-regulated genes,including 5ribosome-related genes.After verification,it was found that they were downstream genes directly regulated by acetylation.The results showed that the M.haplocalyx EO inhibited the acetylation of F.proliferatum histone to inhibit the biosynthesis and function of ribosomes.8.M.haplocalyx EO and carvone can induce the expression of histone deacetylase gene.ConclusionM.haplocalyx EO has a good inhibitory effect on F.proliferatum.It not only can inhibit spore germination and mycelial growth but also can destroy the permeability of the cell membrane of F.proliferatum and reduce the activity of cell wall degrading enzymes.The EO and main components of M.haplocalyx can activate the expression of histone deacetylase gene of F.proliferatum,reduce the acetylation level of histone H3 to inhibit the function of ribosomes,and ultimately lead to the failure of normal growth and development of F.proliferatum to achieve the purpose of antifungal. |
PDF Full Text Request