Studies On Molecular Mechanism Of Tea Plant Against Anthracnose Infection And Assessment Of Freezing Tolerance Of Tea Cultivars

Fungal infection and freezing are typic biotic stress and abiotic stress in tea plant respectively,both of which are getting great attention and concern.The present study focuses on anthracnose and freezing injury in tea plant,which aims at probing the molecular mechanism and regulatory network with regards to resistance to these stresses,and searching for molecular markers associated with resistance so as to facilitate resistant breeding of tea cultivars with marker assisted selection?MAS?.Breeding tea cultivars resistant to anthracnose and freezing injury is considered to be the most effective way to protect tea production against disease and freezing,which owns great scientific significance and practical application in tea industry.Next generation sequencing?NGS?,RNA-seq,dual RNA-seq,gene cloning,high-performance liquid chromatography?HPLC?,real-time quantitative reverse transcriptase PCR?qRT-PCR?,parallel reaction monitoring?PRM?,maximum photochemical efficiency of photosystem II?PSII??Fv/Fm?test,colorimetric test were used in the present study to solve the scientific problems including:1)studying the fungi diversity and pathogenic activity on anthracnose-infected leaves and providing pathogens for further in vivo or in vitro test;2)establishing methods for rapid inoculation of anthracnose,exploiting resistance genes and molecular markers,revealing the interaction relationship between tea plant and Colletotrichum ans also the roles of key genes,proteins and phytohormone in the interaction;3)providing instrumental tools for assessing freezing tolerance of tea cultivars in tea breeding program.The main results are as follows:?1?Fungal diversity test showed healthy leaves had high abundance of Cladosporium and Ramularia,while disease infected leaves had high abundance of Colletotrichum in premetaphase stage and Pseudopestalotiopsis in the late stage with lower abundance of Colletotrichum in the late stage,indicating that Colletotrichum was the main pathogens and Pseudopestalotiopsis increased significantly to make for the formation and enlargement of the disease infexted spots with Colletotrichum in the late stage.?2?The results of pathogen isolation and purification showed the newly anthracnose infected leaves by re-inoculating the primary isolate were the optimum material for isolating target pathogens.By this method,6 out of the obtained 9 strains were Colletotrichum,i.e.,the isolation rate is 67%.Molecular identification confirmed the genus of the 45 isolates and Colletotrichum gloeosporioides or its complex was the dominated pathogen.?3?A de novo transcriptome assembly dataset was generated from healthy and anthracnose-infected leaves on tea cultivars“Longjing-43”?LJ43?and“Zhenong-139”?ZN139?,with 1621 DEGs in LJ43 and 3089 DEGs in ZN139.The GO and KEGG enrichment analysis revealed that the DEGs were highly enriched in catalytic activity,oxidation-reduction,cell-wall reinforcement,plant hormone signal transduction and plant-pathogen interaction.The key genes including ALD1,NPR1 and PR1were cloned.Based on the nucleotide sequence,amino acid sequences of the genes were deduced.Physical and chemical properties and structural models of these proteins were predicted.Further studies by qRT-PCR and HPLC showed that the genes involved in endogenous salicylic acid biosynthesis and accumulation of foliar salicylic acid responsed to anthracnose infection.This study was the first one to provide novel insight in PR1protein and salicylic acid acting as key compounds in the responses to anthracnose infection in tea plant.?4?An improved method by combining wound inoculation with computer image recognition technology was established to assess anthracnose-resistance of tea plant.The results showed that cultivars‘LCS',‘4-52'and‘4-147'were rated as anthracnose-resistance cultivars,while cultivars‘WM1',‘WM3'and‘FZ-0'were anthracnose-susceptible cultivars.Saffron-green staining test revealed that the difference in anthracnose resistance between tea cultivars may be related to cell wall appositions and increasement of lignin.A time-resolved dual transcriptome including 2 tea cultivars?‘LCS'and‘WM3'?with five inoculation time?0 h,12 h,30 h,72 h and 120 h?by Colletotrichum gloeosporioides isolate TJB44 was generated on Illumina HiSeq X Ten platform.Differential expression and enrichment analysis showed that there were 7425DEGs participating in responses to pathogens in total,which were mainly involved in cell wall,secondary metabolites,enzymes,phytohormones,peroxides,signaling,transcription factors,PR proteins,etc.,suggesting that the anthracnose-resistance cultivar may be able to respond to Colletotrichum invasion signals faster than anthracnose-susceptible cultivar in the early stages of infection.Differential expression and enrichment analysis involved in Colletotrichum showed that there were 3697 DEGs involving in the invasion process.A heat map involving the top 20 expression level DEGs showed that the pathogenicity of Colletotrichum was significantly relevant to phytotoxins?like cerato-ulmin,cerato-platanin?,pectin lyase and other enzymes that damage the cell wall,woronin bodies,CAP20 protein and CFEM effectors.PRM protein quantification analysis showed that the level of chitinase?CHIT?,thaumatin-like protein?TLP?,phenylalanine ammonia lyase?PAL?increased significantly,which is consistent with transcriptome data.There was Study showing that these 3 enzymes may be involved in compounds degradation in cell wall by pathogens.The PR1 protein level in healthy leaves in the resistant cultivar‘LCS'was significantly higher than that in the susceptible cultivar‘WM3',while PR1 protein was decreased in the inoculation group.However,PR1 gene was significantly up-regulated in the inoculation group,which might be a feedback response generated by the lack of PR1 protein.The time-resolved dual transcriptome dataset in this study will facilitate to profile gene expression,metabolic networks and host-pathogen interaction associated with tea plant immunity against anthracnose.Numerous DEGs and their encoded proteins need to be further confirmed.?5?Freezing sensitivity index?H?of 47 clonal tea cultivars was investigated after severe freezing winter in 2016.To develop instrumental methods for freezing tolerance assessment,Fv/Fm and leaf color indicator a on the Hunter color scale were determined in frozen group?tea shoots being frozen at-15°C for 2 h and then stood at 20°C for 5h?,with on non-frozen shoots as control.The ratios of control group to frozen group were expressed as R_Fv/Fm and Ra respectively.A linear regression equation was established with H as the dependent variable and R_Fv/Fm or Ra as the independent variable,which was H=60.31-50.09 R_Fv/Fm?p<0.01?and H=30.03-10.82 R_a?p<0.01?respectively.Expression of gene psbA encoding D1 protein in PSII showed that the frezzing tolerant tea cultivars maintained a high expression level of psbA after freezing stress,which is considered to be beneficial to de novo synthesis of D1 protein and sustaining PSII activity.These findings can provide instrumental tools for assessing freezing tolerance of tea cultivars during tea breeding program.