Identification Of Pathogen Caused Tea White Scab Disease And Pathological Characteristics Of Tea Infected By The Pathogen

Tea white scab disease is a fungal disease of tea frequently occurring in high altitude areas every year.The disease has become a complicated,long-unsolvable problem for tea industry due to its increasingly occurrence and damage.To date,systemic studies on the disease’s etiology,regularity of outbreak,and mechanism of pathogenicity are still lacking.In this study,the pathogen of tea white scab disease was identified and found to be a new pathogenic fungus of tea.The pathogen’s biological characteristics were tested,and a series of changes induced by the disease were investigated in physical structure,physiological components,biological molecules and interfoliar microbial community.The major results obtained in this study are shown as bellow:(1)The pathogen of tea white scab disease occurring in Southern China was indentified to be Elsinoe leucospila Bitancoum Jinkins.Two fungal species,Type one and Type two were isolated in 358 samples from Southern China areas including Guangdong,Guangxi and Hunan provinces,but only Type one induced symptoms characteristic of the disease when tested by Koch’s postulation.Sequencing and phylogenetic analyses of ITS,LSU,RPB2 and the 18 S r RNA genes grouped this species and Elsinoe spp.in the same cluster,with high nucleotide identities between them.This finding,together with morphological identification of the fungus,demonstrated for the first time that the pathogen of tea white scab disease occurring in Southern China was E.leucospila.The Type two isolated in this study developed a necrosis spot symptom of leaf in the inoculation test identified as Phoma sp.CPT,which was distinct from that of white scab disease.With regard of its notably faster growth and sporulation relative to E.leucospila,it was considered to be a secondary parasite in the late stage of tea white stab disease.Biological tests showed that E.leucospila had the fastest growth rate,0.1 cm/d,when cultured on PSA medium with sucrose as carbon resource;its optimal nitrogen source was glycine,and other optimum growing conditions as p H7 and 26℃,while the lethal temperature for its hyphae was 40℃.(2)The alterations induced by tea white scab disease in leaf structure,physiological components and defensive enzymes were identified.The alterations induced by tea white scab disease in leaf structure,physiological components and defensive enzymes were identified.After the infection and pathogenesis of tea leaf by E.leucospila,microscopic observation showed that the pathogen presented scattered clumps in the lesion,the cells in the upper and lower epidermis invaded were browned and necrotic,the tea tissues around the invasion site were closely distributed,and the nuclei were enlarged and concentrated in the direction of invasion.The sensory quality evaluation showed that the tea made from diseased leaves tasted stinky and bitter,and the severer the disease was,the stinkier and bitterer the tea tasted.The total amount of amino acids,gamma-aminobutyric acid,serine,alanine and phenylalanine in diseased leaves were positively correlated with the the disease’s occurrence rate.The change trend of EC and ECG content of catechin monomers with higher total flavonoids and oxidative activity was consistent with the occurrence of the disease.Meanwhile,in the disease’s pathogenesis,the POD,PPO,SOD and Chi enzyme activities were all higher than that in the healthy control,while the activities of CAT and PAL increased at 7 dpi and decreased at 14 dpi compared with the blank control.Differential expression analysis of stress resistance-related genes in diseased tea at the transcriptomic level.A total of 65 and 139 differentially expressed genes(DEGs)were detected via transcriptome sequencing and analysis at 7 dpi and 14 dpi respectively,in diseased tea leaves.Two genes,the cytochrome P450 gene and a novel gene,were found being expressed in the whole post-infection period.GO analysis showed that the cell components,molecular functions and biological metabolic processes in the host were significantly altered after the disease infection.These DEGs mainly involve in biosynthesis,metabolic pathways,sulfur metabolism,flavonoid biosynthesis,photosynthesis antennal protein and endoplasmic reticulum protein processing of secondary metabolites,etc.At the same time,we identified gens related to stress resistance were mainly 9 up-regulated genes encoding Hsp90.1,Hsp70 and TT4,etc.genes.Also,E.leucospila was found closely related to the genes in diseased tea leaves.In addition,with Beta-actin taken as the internal reference genes.the results of Q-PCR were consistent with the transcriptome results,which were performed to verify the two differentially expressed genes encoding Hsp90.1 and F3 H.(3)Analysis of tea white scab disease induced differences in phyllospheric microorganism populations.High throughput sequencing showed that the phyllospheric microorganism communities in infected samples were notably altered.With increasing disease severity,the overall OTUs,differential OTUs and diversity of the communities increased.In this study,the most significantly affected was the abundance of the populations in phylum Proteobacteria,whose relative abundance was 90.25% in healthy tea leaves,but only 85.51% and 73.5% in moderately(11%～50% of leaf area affected)and severely(over 80% of leaf area affected)diseased samples,respectively.In addition,the relative abundances of bacteria from genera Methanobacillus,Hymenobacillus,Strelitziana albiziae,Cercospora apii and Elsinoaceaewere positively correlated with disease grade,while that of rexeniella and oligotrophomonas was in contrary.It was indicated that the phyllospheric microorganisms from genera Ethanoligenens,Taibaiella,Rubinisphaera,Microbacter and Phaselicystis were specific in the severest diseased samples.This suggested that the phyllospheric microorganism community may play an important role in occurrence of tea white scab disease.In summary,the determination of the pathogen of tea white scab disease in this study provides fundamental information for understanding the occurrence regularity of this disease and optimizing the technological strategy of disease prevention and control;and the obtained results from further investigations of the tea white scab disease induced changes in pathological phenotypes,physiological components,enzymatic activities,transcriptome and phyllospheric bacterial populations are of both theoretical and practical importance,because these results may provide a scientific basis for disease prevention and control strategy development and ensures safety of tea production.