Effects Of Rhododendron Delavayi Petals On Four Nonspecific Toxin Of Alternaria Tenuissima

Guizhou Baili Rhododendron nature reserve is a national ecotourism demonstration area.It is the only nature reserve in China to protect Alpine Rhododendron.It is an important Rhododendron plant gene bank in China.However,the occurrence of diseases in the nature reserve is more serious in recent years,among which petal blight of Rhododendron is a disease that seriously endangers the development of the nature reserve.Petal blight causes a variety of rhododendrons to become smaller,the surface of petals loses luster,water stains patches or brown spots appear,the petals gradually soften until rot,and the flowering period is seriously shortened.Over the years,there have been few reports on petal diseases in the nature reserve.In order to ensure its healthy development.The sample petal blight of Rhododendron delavayi,the main species in Guizhou Baili Rhododendron nature reserve,were used as experimental materials.The pathogens of petal blight disease of Rhododendron delavayi were isolated and identified by microbial isolation and culture,Koch’s rule verification,morphological observation and phylogenetic analysis.The results showed that four strains（MR5,MR6,MR9 and MR10）were isolated from the diseased tissues.Through the determination of the pathogenicity and toxin producing ability of four pathogenic bacteria.Finally,Alternaria tenuissima（MR9）was selected as the object for further research.Six different media were prepared by using different amounts of R.delavayi petals,including PDA,PDA,5g-MY-PDA,50g-MY-PDA,100g-MY-PDA,200g-MY-PDA,200g-MY.The common PDA medium was used as the control group.The toxin content of A.tenuissima after treatment was determined,in which alternariol（AOH）,alternariol monomethyl ether（AME）,tenuazonic acid（Te A）and alternane（ALT）.Referring to the results,200 g R.delavayi petals potato glucose agar medium was selected as the treatment group and ordinary PDA medium as the control group.Transcriptome sequencing and proteome sequencing were used to analyze,in order to find the relevant pathogenic genes of MR9 and provide scientific basis for the prevention and control of A.tenuissima disease.The main results of this study are as follows:1.The pathogen of Rhododendron petal blight wilt collected from Baili Rhododendron Nature Reserve in Guizhou was isolated and identified.The pathogenicity test results showed that the pathogens could infect Rhododendron petal blight and cause petal wilt disease consistent with the symptoms in the forest.Observation of morphological characteristics of pathogens and use of internal transcribed spacer（ITS）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）and 1-α translation elongation factor 1-alpha（TEF1）,Alternaria major allergen gene（Alt a 1）and RNA polymerase second largest subunit（RPB2）.The results showed that the four pathogens causing Rhododendron petal blight were Alternaria tenuissima.2.Through the pathogenicity determination of four pathogens,MR9 strain was selected as the research object of the next experiment.MR9 strains were inoculated on six different media.The effects on the growth of MR9 strain were observed,mainly in the diameter of colony and the density of mycelium.The results showed that the growth rate of MR9 increased with the increase of the amount of R.delavayi petals.However,the density of MR9 strain decreased gradually with the increase of the number of petals.The results showed that R.delavayi petals promoted the growth of MR9 strain,but inhibited the density of colony.3.High performance liquid chromatography（HPLC）was used to determine the toxin production of MR9 strain under six different culture conditions.The results showed that the contents of four nonspecific toxin decreased significantly compared with that on PDA medium,and the toxin production ability of MR9 decreased with the increase of the addition of R.delavayi petals,and the effect on AOH and AME toxins was the most obvious.In addition,the activity of Alternaria toxin bioassay solution was determined.The results showed that the petals of healthy R.delavayi inoculated with Alternaria toxin bioassay solution appeared brown spots,and gradually expanded with the extension of infection time.The pathogenicity of the Alternaria toxin bioassay also weakened with the increase of the number of petals.The results were consistent with the results of the toxin production of MR9 strain on six different media,indicating that the toxin production ability of Alternaria toxins were closely related to its disease treatment ability.4.According to the results of the effects of six different media on the virulence of MR9 strain,PDA was selected as the control group and200g-MY-PDA as the treatment group for combined analysis of transcriptome and proteome sequencing.The results of transcriptome sequencing,gene annotation,differential expression gene screening,GO and KEGG correlation analysis showed that the genes of MR9 treated by R.delavayi petals were significantly down regulated in the process of metabolite synthesis,antioxidant system and other related pathways,and the differentially expressed genes related to non-ribosomal peptide synthetase were significantly changed,including AALT＿g10556,AALT＿g4616,AALT＿g6627,AALT＿g869,AALT＿g9403,and seven differentially expressed genes（AALT＿g8532,AALT＿g9032,AALT＿g10361,AALT＿g7484,AALT＿g9253,AALT＿g3816,AALT＿g789）were found to be involved in MAPK signaling pathway.It has been reported that NRPS and MAPK signaling pathways are closely related to the pathogenicity of A.tenuissima.In addition,by analyzing the results of proteome sequencing,it is found that the differentially expressed proteins are mainly enriched in detoxification,antioxidant reduction process and macromolecular metabolite synthesis.The results of proteome sequencing are similar to those of transcriptome sequencing,mainly enriched in antioxidant process and metabolite synthesis.The results showed that R.delavayi petals could inhibit the expression of pathogenic genes of A.tenuissima,reduce the sensitivity of A.tenuissima to the production of reactive oxygen species,and then reduce the infection ability of A.tenuissima to the host,and even inhibit the synthesis of Alternaria toxin.Through the combined analysis of proteome and transcriptome,it is found that the pathway significantly enriched in both proteome and transcriptome is the metabolism of xenobiotics by cytochrome P450,in which the protein / gene annotated into this pathway is AALT＿g4490/OWY51204.1（glutathione-S-transferase）.Glutathione（GST）plays an important role in scavenging reactive oxygen species.In this process,glutathione-S-transferase plays an important catalytic role.The results of combined analysis proved that the pathogenicity of A.tenuissima was closely related to the genes related to antioxidant system.