Tea Leaf Spot Resistance Comparison And Metabolic Regulation Of Anthocyanin From Tea Plant

Tea is a traditional drink in China.In recent years,it has become popular around the world.Its yield and quality depend on the growth and development of tea plants in various environments.Nowadays,leaf diseases caused by fungal infection are one of the main causes of tea quality and yield reduction,which brings great pressure to the production of high-quality tea.Tea leaf spot is a common leaf fungal disease in tea plants.At present,the main means of controlling tea leaf spot is the use of chemical pesticides.However,the prevention and control of chemical pesticides bring corresponding pressure to the environment and easily lead to pathogen resistance.At present,bioactive plant-derived natural products have been explored as an effective method to replace chemical fungicides.As a natural plant-derived flavonoid compound,anthocyanin has antibacterial biological activity and has the potential to be developed as a new bacteriostatic agent,but its anabolism regulation remains to be studied.1.In this study,four fungi were isolated and purified from the surface of susceptible leaves by PDA medium,named Tj-A,Tj-B,Tj-C and Tj-D.Among them,Tj-D had the strongest pathogenicity,which was verified as the pathogen of tea leaf spot by Koch’s rule.After morphological identification and sequence determination,it was identified as Lasiodiplodia theobromae.This is the first time that the pathogen was isolated from the susceptible leaves of tea leaf spot in Hangzhou tea garden.The five tea varieties of Ziyalongjing,Zhongcha 108,Huangjinya,Baiye 1 and Zijuan were infected by Tj-D,and the area infected at the same time showed a negative correlation with the anthocyanin content.The Ziya Longjing variety with the highest anthocyanin content and the least susceptibility to tea leaf spot was selected.Different color leaves on both sides were obtained by different light induction,and the infection test was carried out again.The results showed that the purple leaves on the shading side were more resistant to tea leaf spot than the green leaves on the unshaded side.Anthocyanin has a positive correlation with tea plant resistance2.The leaves with different colors on both sides obtained by different light induction in the above experiments were used as materials for analysis from the metabolomics and transcriptome layers.The results of metabolomics showed that the content of anthocyanins in the shaded side(purple leaves)was significantly higher than that in the unshaded side(green leaves).A total of 27 significantly different metabolites were screened between the two groups,of which 24 were anthocyanins.Except for delphinidin-3-O-rutinoside,delphinidin-3-O-rutinoside-5-O-glucoside,peonidin-3-O-5-O-(6-O-p-coumaroyl)-diglucoside,2023the remaining 21 anthocyanins were higher in purple leaves.The relative increase of pelargonidin-3-O-galactoside in purple leaves was the most significant,which was 51.24 times higher than that in green leaves.The absolute increase of cyanidin-3-O-galactoside and delphinidin-3-O-galactoside was the most significant,which increased by 1128.20 μg/g and994.37 μg/g respectively compared with green leaves.It is speculated that anthocyanin galactoside plays a certain role in the disease resistance of tea plants.3.The results of transcriptome showed that the differentially expressed genes of Ziyalongjing in different treatment groups were mainly enriched in photosynthetic pathway and secondary metabolite pathway,and some of the differentially expressed genes were closely related to flavonoid biosynthesis pathway and anthocyanin biosynthesis pathway.The expression level of FLS gene in purple leaves was generally lower than that in green leaves,indicating that the trend of dihydroflavonols in purple leaves to flavonols was slower than that in green leaves,and thus more flowed to the anthocyanin synthesis branch.The significantly differentially expressed genes of the UGT family with higher expression in the shading P group mostly acted on flavonoids including anthocyanins,indicating that anthocyanin-related UGT genes showed a positive response to shading treatment.Under shading conditions,the expression of UGT family genes with anthocyanin as the substrate increased,which was conducive to the accumulation of anthocyanin in tea plants.This result was consistent with the increase of anthocyanin and its glycoside content analyzed by metabolomics.4.The key regulatory genes CSS0020068 and CSS0047476 of anthocyanin metabolism were selected for cloning and subcellular localization analysis.The cloning results clarified the specific sequence information of the gene.In addition,35 S : GFP-CSS0020068 localization and 35 S : GFP-CSS0047476 fluorescence did not overlap with chloroplast fluorescence by Agrobacterium-mediated tobacco transient expression test.The results help to strengthen our understanding of these two UGT genes at the molecular and cellular levels,and provide a basis for further verification of gene function.The development of related work provides a theoretical basis for the directional regulation of anthocyanin synthesis and the development of biological pesticides.In this paper,the resistance analysis of tea anthocyanin to tea leaf spot disease was explored.The leaves with different resistance were analyzed from the metabolomics and transcriptome levels,and two of the key regulatory genes of anthocyanin metabolism were selected from the multi-omics screening for cloning and subcellular localization analysis.The conclusions obtained provide a theoretical basis for better development and utilization of natural plant-derived flavonoids,and provide a theoretical basis for biological control of tea gardens.