【作者】 周凌云；

【导师】 王振中；

【作者基本信息】 华南农业大学 ， 植物病理学， 2019， 博士

【摘要】 茶白星病(Tea white scab disease)是我国高海拔茶区常发真菌病害,近年发生面积及其为害程度呈上升趋势,对我国茶产业发展构成重大威胁。目前对于该病的病原、发生规律和致病机理等均缺乏系统研究。本研究对我国茶白星病进行病原鉴定及病菌生物学特性测定,进而对茶白星病引致的茶叶细胞结构、生理生化、转录组及叶际微生物群落的变化进行了系统研究。主要结果如下:1.鉴定了中国南部茶区茶白星病的病原为茶痂囊腔菌(Elsinoe leucospila Bitancoum Jinkins)。从中国南部广东、广西、湖南3省358份茶白星病样中分离到两种主要真菌T1和T2,其中T1回接引致茶白星病症状,PCR扩增其ITS、LSU、RPB2与18S r RNA编码基因片段,序列测定和比对发现,T1菌株的上述4个基因核苷酸序列与Elsinoe spp.同源率最高,并结合形态学测定,将中国南部茶区茶白星病的病原鉴定为Elsinoe leucospila Bitancoum Jinkins。T2的ITS、LSU、RPB2与18S r RNA基因序列与Phoma spp.同源率最高,初步鉴定为Phoma sp.CPT,但该菌在回接试验中不引致茶白星病症状,因其生长与产孢速度明显快于E.leucospila,极易从病组织中分离获得,推测其为茶白星病发生后期的次寄生菌。生物学特性测定结果表明,E.leucospila在以蔗糖为碳源的PSA培养基上生长相对最快,但生长速率也仅为0.10 cm/d,该菌菌丝生长最适氮源为甘氨酸,最适酸碱度为p H7.0,最适温度为26℃,菌丝致死温度为40℃。2.测定了茶白星病引致茶叶病理特征动态变化。茶白星病菌侵染茶叶后,在侵入点附近形成显微镜下可见的菌丝簇,引致叶片上下表皮细胞褐变坏死,并诱导叶肉细胞聚集在侵入点附近,其中部分细胞的细胞核膨大。感病茶叶制成的饮品味苦具腥气,病情越重苦味和腥气越浓。感病叶片氨基酸总量、r-氨基丁酸、丝氨酸、丙氨酸、苯丙氨酸含量显著增加,总黄酮及儿茶素单体EC与ECG含量也有所增加,并均与叶片病情呈正相关。在病程发展过程中,在感病叶片中的POD、PPO、SOD及几丁质酶一直高于其在健康叶片中的酶活,而CAT和PAL酶活在病程第7 d时升高,而在第14 d时下降。3.比较了茶白星病引致的叶片转录组差异表达。转录组测定表明,茶白星病菌侵染7 d和14 d时,发病叶片分别有65与139个基因出现差异表达,并发现侵染病变后一直差异表达的2个基因,分别为细胞色素P450基因与1个新基因。富集分析表明差异表达基因主要富集在次生代谢产物的生物合成、代谢途径、硫代谢、类黄酮生物合成、光合作用-触角蛋白质及内质网蛋白质加工等途径。同时,鉴定到抗逆相关的基因主要为编码Hsp90.1、Hsp70及TT4等9个上调相关基因,显示这些基因与E.leucospila菌引致茶叶发病密切相关。此外,以β-actin为内参基因,Q-PCR验证编码Hsp90.1与F3H的2个上调差异表达基因,发现与转录组结果具有一致性。4.揭示了茶白星病引致的叶际微生物群落变化特征。高通量测序表明,受茶白星病菌侵染后,茶叶叶际微生物群落发生了明显的变化,随着病情等级升高,叶际微生物群落整体OTUs、差异OTUs及其多样性均上升,而变形菌门丰度在健康叶片中高达90.25%,在病情中等(病斑面积占叶面积的11%～50%)和严重(病斑面积占叶面积的80%以上)的叶际其相对丰度分别为85.51%和73.53%。此外,甲醇杆菌、膜杆菌、合欢横断孢、褐尾孢菌与痂囊腔菌等微生物的相对丰度与病情等级呈正相关,而雷氏菌与寡养单胞菌、欧洲硫菌属等则相反。茶白星病发病最重的叶际特有菌主要有Ethanoligenens、Taibaiella、Rubinisphaera、Microbacter与Phaselicystis。表明叶际微生物群落可能在茶白星病的发生过程中发挥了重要作用。综上所述,本研究首次明确了我国南方茶区茶白星病的病原,为该病害发生规律研究和防控技术研发提供了基础,较全面地解析了茶白星病引致的茶叶组织结构、生化成分、转录组及叶际微生物的变化,深化了对茶白星病为害及其致病机理的认识,具有一定的理论意义和应用价值。更多还原

【Abstract】 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.更多还原