| Flue-cured tobacco is an important cash crop in China.Seedling is the primary link of tobacco production.Selecting strong seedling and transplanting is the basis for flue-cured tobacco to grow well in the field.Some studies have shown that the Exobasidium can produce plant growth regulating substances and regulate the growth of non-host plants to some extent.Therefore,pathogen strains were isolated by tissue suspension method and purified.The basidia and basidiospores morphology were observed under light microscopy and scanning electron microscopy,morphology of colonies on PDA were observed under stereo-fluorescence microscope.The pathogen was identified by morphological characteristics,r DNA-ITS and r DNA-LSU polygenetic phylogenetic analysis.The contents of plant growth regulating substances,amino acids and vitamins in the metabolites of pathogenic fungi were analyzed by liquid mass spectrometry(LC-MS/MS).To investigate different concentrations [the original solution,1/10 original solution,1/50 original solution,1/100 original solution] of E.japonicum liquid culture on growth and key enzyme of carbon and nitrogen metabolism of flue-cured tobacco seedlings were studied using 'K326' as the test material and transcriptome analysis was conducted to explore the regulation mechanism of Exobasidium liquid culture on flue-cured tobacco seedlings that expected to provide theoretical basis for the production of high-quality tobacco.The results are as follows:1.The results showed that leaf swelling in R.pulchrum was caused by E.japonicum in Fenghuangshan Park,Guiyang City,Guizhou Province.2.Untargeted metabolomics analysis based on liquid-mass spectrometry(LC-MS/MS)technique revealed seven plant growth substances in the culture medium of E.japonicum,including indole acetic acid(IAA),indole butyric acid(IBA),naphthalene acetic acid(NAA),abscisic acid(ABA),kinetin(KT),jasmonic acid(JA)and Spermidine.Contains Serine,Lysine,Glutamic acid,Histidine,Phenylalanine,Tryptophan,Valine and Arginine,Threonic acid,Tyrosine and Aspartic acid 14 amino acids,including acid,Glycylproline,Homocysteine and Ornithine;Contains 5 kinds of vitamins such as vitamin B2,vitamin B3,vitamin B5,vitamin B6 and vitamin C.3.The effects of different concentrations of E.japonicum liquid culture on the growth of flue-cured tobacco seedlings and the production of key enzymes in carbon and nitrogen metabolism were different.On the third day after treatment,1/50 original solution had the most significant promoting effect on root activity of flue-cured tobacco seedlings,and the contents of chlorophyll a,chlorophyll b and carotenoids in leaves were significantly increased after the original solution treatment.On the 7th day after treatment,the activities of five enzymes(NR,GS,SSS,SPS and INV)in flue-cured tobacco seedlings reached the peak value,and were significantly higher than those in the control group.On the 11 th day after treatment,leaf length,leaf width,leaf area and plant height of flue-cured tobacco seedlings treated with 1/100 solution were significantly higher than those of the control group.Considering the effects of culture medium of E.japonicum on the growth and physiological indexes of flue-cured tobacco seedlings,1/100 original solution had the best effect,and 1/50 original solution had the second effect.4.In this study,transcriptome sequencing was performed on flue-cured tobacco seedlings treated with 1/100 original solution and 3 d and 11 d,among which the number of DEGs was the largest in the CK treated and 1/100 original solution with 3d.DEGs are mainly involved in biological processes such as metabolic process,cellular process,tissue process,cell and cell component composition.Metabolic pathways,biosynthesis of secondary metabolites,plant hormone signal transduction and carbon metabolism were the main pathways for the enrichment of differential genes.At 3 days after treatment,AUX1,TIR1,JAZ,NPR1 and TGA genes in plant hormone signal transducting pathway were significantly up-regulated,while JAR1,Co I1 and MYC2 were significantly down-regulated.AUX/IAA is auxin induction factor,ARF is auxin response,AUX/IAA genes is up-regulated and ARF genes is down-regulated.Jasmonic acid-amino acid synthase(JAR1),mutant insensitive to coronavirin(COI1),and signal transduction factor(MYC2)were all significantly down-regulated,while inhibitory protein(JAZ)was significantly up-regulated,negatively regulating JA synthesis as a whole,both NPR1 and TGA genes were significantly up-regulated in the SA synthesis pathway,which had a significant promoting effect.The genes of most of the enzymes involved in the carbon metabolism pathway,such as PRK,Rubisco,GAPDH and TPI,were significantly up-regulated.5.Different concentrations of E.japonicum liquid culture had different effects on the growth and antioxidant metabolism of the upper three leaves of flue-cured tobacco:Original solution had a certain regulation effect on soluble protein,MDA content and POD activity in the upper leaves of flue-cured tobacco.Compared with CK,soluble protein content in the first,second and third leaves of flue-cured tobacco was increased by 0.24 U·g-1?0.04 U·g-1 and 0.02 U·g-1,respectively.MDA content in the first leaf was significantly decreased by 39.80% compared with CK,and POD activity in the third leaf was significantly increased by 50.32% compared with control.The original solution also promoted the openness of the first leaf,and the opening degree increased by 0.07 compared with the control.1/10 original solution had a great effect on MDA content and CAT activity in the upper leaves of flue-cured tobacco.MDA content in the second leaf of flue-cured tobacco was significantly reduced by 28.25% compared with the control.CAT activity in the second and third leaves of flue-cured tobacco was increased by 18.53% and 48.56% compared with CK,respectively.At the same time,1/10 original solution can obviously promote the growth of the second leaf.The 1/50 original solution had different effects on H2O2 content,MDA content,SOD,POD and CAT activities in the upper leaves of flue-cured tobacco.H2O2 content in the first,second and third leaves of flue-cured tobacco was reduced by 462.38 mmol·gprot-1 ? 206.73 mmol·gprot-1 ? 845.42mmol·gprot-1 compared with the control,respectively.The SOD activity of the second and third leaves of flue-cured tobacco was increased by 103.65% and 96.95%,and the POD activity was increased by 82.33% and 63.62%,respectively,compared with that of CK.The CAT activity of the third leaf of flue-cured tobacco was increased by 29.25% and the MDA content was decreased by 39.16% compared with CK,respectively.The openness degree of the first leaf in the upper part of flue-cured tobacco was significantly increased by 0.1 and the width of the third leaf was significantly increased by 4.62 cm compared with CK.Considering the effects of culture medium of E.japonicum on the growth of upper leaves of flue-cured tobacco and the activities of antioxidant metabolic enzymes,1/50 original solution had the best effect,and 1/10 original solution had the second effect. |
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