| Tea plant(Camellia sinensis) is one of the most economically valuable crops in the world. Anthracnose, caused by Colletotrichum spp.,can invadetea leaves and cause serious yield losses.Developing anthracnose-resistant tea cultivars depending on its native resistance is the most effective and economic approach to controlanthracnose in practice.At present, research on the diversity and geographical distributionof Colletotrichumin China remainlimited. Moreover, the roles of secondary metabolites of tea plant in resistance to anthracnose are unclear.In our research, we systematicallystudythe diversityof Colletotrichum species causing anthracnose of tea plantsin China, and analyze the interaction relationship between Colletotrichum and tea plants, using the resistant Ca. sinensis cv. Zhongcha108(ZC108) and susceptible Ca. sinensis cv. Longjing43(LJ43) as experimental materials. The main results were summarized as follows:1. In this study, 106 Colletotrichum isolates were collected from diseased leaves of Ca. sinensis cultivatedin the 15 main tea production provinces in China. Multi-locus(ACT, CAL, CHS-1, GAPDH, GS and ITS) phylogenetic analysis coupled with morphological identification showed that the collected isolates belonged to 11 species in five speices complexs, including six known species(C. camelliae, C. cliviae, C. fioriniae, C. fructicola, C. karstii, C. siamense), three new record species(C. aenigma, C. endophytica, C. truncatum), one novel species(C. wuxiense), and one undistinguishable strain, herein described as Colletotrichum sp. Of these species, the numbers of species in C. gloeosporioides species complex were more than the other species complex(7/11), C. camelliae and C. fructicola were the dominant species causing anthracnose in Ca. sinensis. In addition, our study provided further evidence that phylogenetic analysis using a combination of Ap Mat and GS sequences can be used to effectively resolve the taxonomic relationship of the C. gloeosporioides species complex. Finally, pathogenicity tests suggested that C. camelliae, C. aenigma, C. endophytica C. fructicola and C. truncatum are more invasive than another six speices(C. cliviae, C. fioriniae, C. karstii, Colletotrichum sp., C. siamenseand C. wuxiense)after the inoculation on the leaves of LJ43.2. The germination and infection processes of the conidia of C.fructicolaon different resistant tea leaves were detected by tissueclearing. The results demonstrated that the conidia of C.fructicolagerminated in different resistant tea leaves at 12 hours after inoculation,and appressoria developed at the same time, and the numbers of germinated conidia and appressoria on resistant ZC108 were less than that on susceptible LJ43; at 96 hours after inoculation,the growth and development of C.fructicolaon ZC108 were obvious delayed during inoculation time course.3. The production of H2O2 and HR was detected using DAB and trypan blue staining respectively in the tea leaves inoculated by C.fructicola. The results demonstrated that H2O2 generation and HR reaction was observed on both of and LJ43 leaves, but the generated time of those reactions on ZC108 was earlier than LJ43 for 24 hours, and the reaction was more intensive. These results demonstrated that H2O2 and HR play an important role in defending the anthracnose.4. Herein we compared the contents of total phenolic, catechins and caffeine in two cultivars with different resistance to anthracnose during Colletotrichum fructicola infection. The(-)-epigallocatechin-3-gallate(EGCG),(+)-catechin(C), caffeine, and critical regulatory genes were induced in C. fructicola-resistant tissues.In vitro antifungal tests showed that caffeine more strongly inhibitedmycelial growth than tea polyphenols and catechins. Both electron microscopy and bioactivity analysis results showed that caffeine can affect mycelial cell walls and plasma membranes. Throughpromoter sequencesanalysis, a number of stress response-related cis-acting elements were identified in S-adenosylmethionine synthetase and tea caffeine synthase. These results demonstrated that(-)-EGCG,(+)-C, and caffeine may be involved in resistance of tea plants to anthracnose.5.Transcriptomic analysis of tea leaves from resistant ZC108 and susceptible LJ43 at 0, 24 h and 72 h after inoculation with C.fructicolawere detected by RNA-seq. The results showed that at least 11.75 G high quality clean bases were obtained from each sample; a total of 59,336 and 35,310 unigenes were producedfrom ZC108 and LJ43 respectivelyafter independent assembly using Trinity; based on At No PDB database, the 35,935 and 35,310 unigenes were annotated respectively and sequentially 19,718 and 15,848 differentially expressed genes were identified by RSEM; these differentially expressed genes(DEGs) were functionally classified based on GO. The results indicated that most genes were involved in responses to stress in the two cultivars, but a larger number of DEGs from ZC108 were enriched in each pathway; the results of KEGG showed that a number of DEGs from ZC108 were associated with the biosynthesis of phytohormone and caffeine, but the DEGs from LJ43 were mainly enriched in ribosome pathway. Above all, we speculated that the biosynthesis of phytohormone and caffeine were involved in response to anthracnosein tea plants. |
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