The Active Components And Their Pathogenic Mechanism Of Toxins Produced By Rhizoctonia Solani From Tobacco Target Spot Disease

Tobacco target spot disease is a new type of leaf spot caused by Rhizoctonia solani found in China in recent years,resulting in huge economic losses.The pathogen R.solani can cause pathogenic effects by producing toxins.The fermentation conditions,the active components and pathogenic mechanism of the toxins produced by R.solani were studied in this paper.The results are as follows:1.More than 60 samples of tobacco target spot disease in Puer and Lincang tobacco planting areas in Yunnan were collected,and 68 strains were obtained by conventional tissue separation.A significant positive correlation was found between the strains of different pathogenic strains of tobacco target stains and the stronger the virulence of the strains,and the stronger the virulence of the strains.The strong pathogenic strain SJ-8 was selected as the test material,and the fermentation conditions were preliminarily screened from the variety of fermentation liquid,fermentation time,fermentation temperature,pH value,light condition,fermentation mode,inoculation quantity and culture volume.From the experimental data,we can see that the improved Rhichard culture solution,28?,7 days of culture,7 pH value,light and dark alternation,interval 6h oscillation fermentation liquid,10%inoculation volume percentage and culture volume of 100ml are the best cultivation conditions for fermentation broth.2.TLC and HPLC were used to isolate and purify the toxins of R.solani.The toxin structure was identified by IR,MS,elemental analysis,and NMR.The results showed that crude toxin was obtained through the best deployment of 95%ethanol:concentrated ammonia:ethyl acetate=10:1:1?TLC?.This was further purified by preparative HPLC to give fraction I and fraction V of the protoxin with greater pathogenic activity.The structures of toxin I and V were identified by IR,elemental analysis,mass spectrometry and NMR spectroscopy.The toxin component I was detected by infrared spectroscopy and its structure was found to contain mono-substituted benzene,two alkyl ether?ROR'?,+P-Ar,-CH₂-,and-OCH₃structures.The molecular weight of the phosphate cation was 307 by elemental analysis and mass spectrometry.Combined with the results of nuclear magnetic resonance ¹H-NMR analysis,¹³C-NMR and various two-dimensional NMR spectra,the final analysis was that the toxin component I was methoxymethyl3-phenylphosphonium chloride(C₂₀H₂₀ClOP).The toxin component V was detected by infrared spectroscopy and its structure was found to contain carboxylic acid?-COOH?,1,3-disubstituted benzene,aralkyl ether?ArOR?,-CH₂-,and-OCH₃structures.According to the results of elemental analysis and mass spectrometry HRMS,the quality of[M-H]-peak was 165.0546.Combined with ¹H-NMR,¹³C-NMR and various two-dimensional NMR spectra,the toxin component V was identified as 3-methoxyphenylacetic acid(C₉H₁₀O₃).3.In order to determine the pathogenic mechanism of toxin of R.solani,the effects of 3-methoxy phenylacetic acid and methoxy methyl 3-phenyl chloride toxin on the metabolism of active oxygen,the content of chlorophyll and the ultrastructure of leaf cells were studied.The results of reactive oxygen species test showed that tobacco leaves were treated with mixed components of two toxins and two toxins,which stimulated the leaves to produce defensive reactions.The active oxygen metabolism in leaves increased,and the rate of O^2-production and H₂O₂ production varied greatly.The result of chlorophyll content test showed that the content of chlorophyll of tobacco leaves after treated for 3 days with 3-methoxyphenylacetic acid and methoxymethyl 3-phenyl chlorophosphonotoxin respectively decreased.And the percent reduction in chlorophyll content increases as the toxin concentration increases.Electron microscopy observation showed that the number of mitochondrial ridges in mesophyll cells decreased and endoplasmic reticulum ruptured when treated with 3-methoxy phenylacetic acid toxin 12h.After treatment with 36h,the lamellar structure of chloroplasts showed partial cleavage,and mitochondria began to split,and the ridge disappeared.At 48h,chloroplast membrane,matrix and mitochondria disappeared completely.Only a few lamellar structures remained in some chloroplasts.After treatment of 12h with methoxy methyl 3-phenyl phosphonium chloride,the mitochondrial ridge became coarser,and the local lamellar structure of chloroplast began to lysis.After treatment with 36h,mitochondria appeared vacuolated and the number of ridges decreased and lysis,and the lamellar structure of chloroplasts almost all cracked.At 48h,the cytoplasm wall was separated seriously,and the chloroplast membrane,lamellar structure and mitochondria disappeared completely.