中国茶树炭疽菌系统发育学研究及茶树咖啡碱抗炭疽病的作用

发布时间：2018-04-21 11:08

本文选题：茶树 + 炭疽病　；参考：《西北农林科技大学》2016年博士论文

【摘要】：茶树(Camellia sinensis)是世界重要的经济林木之一。由炭疽菌(Colletotrichum spp.)引起的炭疽病可以危害茶树叶部组织,严重影响茶叶产量。利用茶树自身抗病资源,培育抗病新品种是控制茶树炭疽病最经济、有效的措施。目前,关于我国茶树炭疽菌种类及其地理分布尚不清晰。同时,茶树次级代谢物对抗炭疽病的作用也不明确。本文系统研究了我国茶树炭疽菌的生物多样性,并以茶树抗病品种中茶108(ZC108)和感病品种龙井43(LJ43)作为试验材料,对炭疽菌-茶树互作关系进行了较为深入的研究。主要结果如下:1.从我国15个省(市、自治区)的茶叶主产区多个茶树品种病叶上共分离获得106株炭疽菌,基于多基因系统发育学(ACT、CAL、CHS-1、GAPDH、GS和ITS)和形态学分析,发现这些菌株分别归属于C.acutatum、C.boninense、C.cliviae、C.gloeosporioides和C.truncatum共5个复合种中的11个种,包括6个已知种(C.camelliae、C.cliviae、C.fioriniae、C.fructicola、C.karstii和C.siamense),3个新记录种(C.aenigma、C.endophytica和C.truncatum),1个新种(C.wuxiense)和1个未确定种(Colletotrichum sp.)。C.gloeosporioides复合种的种类最多(7/11),其中C.camelliae和C.fructicola为我国茶树优势致病种。同时,本研究进一步验证了利用ApMat和GS基因快速识别C.gloeosporioides复合种种间关系的方法,结果表明此方法并不能将C.siamense和个别种完全区分,但其依然是快速而有效判断炭疽菌种级关系的重要手段。致病性测试结果表明,C.camelliae、C.aenigma、C.endophytica、C.fructicola和C.truncatum可使LJ43叶片致病,而其他6种(C.cliviae、C.fioriniae、C.karstii、Colletotrichum sp.、C.siamense和C.wuxiense)接种后未发病。2.利用组织透明法观察炭疽菌C.fructicola在不同抗性品种上的萌发侵染情况。结果表明,接种12 h后,炭疽菌C.fructicola在两个品种上均已开始萌发,并形成附着胞,但在ZC108叶片上的孢子萌发数量和附着胞数量明显少于LJ43;接种96h后,炭疽菌C.fructicola在抗病品种上的生长发育明显延迟。3.利用二联基联苯胺和台盼蓝染色法,观察不同抗性品种与炭疽菌C.fructicola互作中H2O2和过敏性坏死(HR)的作用。结果表明,在接种炭疽菌后,两个品种叶片均出现不同程度的H2O2积累和HR反应,但抗病品种比感病品种提前24h响应,并且抗病品种叶片的反应程度比感病品种强,表明活性氧和HR在茶树抗炭疽病过程中发挥着重要作用。4.分别对ZC108和LJ43接种炭疽菌C.fructicola,测定叶片多酚总量、儿茶素类和咖啡碱含量及其相关合成基因的变化。结果表明,(-)-EGCG,(+)-C和咖啡碱含量及其关键基因的表达受到炭疽菌C.fructicola诱导升高;室内抑菌试验结果表明,咖啡碱的抑菌效果要优于茶多酚和儿茶素;电镜观察结果和生理活性测定结果表明,咖啡碱能破坏菌丝的细胞壁和原生质膜;对茶树咖啡碱合成关键基因TCS1和SAMS启动子序列进行分析发现,启动子区域含有多个与植物抗逆性相关的顺式作用元件。综上所述,(-)-EGCG、(+)-C和咖啡碱在茶树抗病过程中发挥了重要作用。5.分别以ZC108和LJ43叶片为材料,对接种炭疽菌C.fructicola后0、24和72h的叶片样品进行转录组测序。结果表明,每个样品测序均获得至少11.75 G clean base;利用Trinity法对2个品种进行独立组装,ZC108共获得59,336条unigenes,LJ43共获得35,310条unigenes;将所有unigenes序列与数据库AtNoPDB比对后进行功能信息注释,ZC108共注释35,935条unigenes,LJ43共注释35,310条unigenes;采用RSEM法筛选差异表达基因,ZC108共获得19,718条差异表达基因,LJ43获得15,848条;对差异表达基因进行GO生物功能分析发现,两个品种的差异表达基因富集到与抗病有关的功能中,但ZC108各通路富集的基因数明显多于LJ43。对差异表达基因进行KEGG富集分析发现,ZC108的差异表达基因主要富集在植物激素合成和咖啡碱代谢通路中,而LJ43主要富集在核糖体通路。以上结果表明植物激素和咖啡碱合成代谢与茶树抗炭疽病有关。
[Abstract]:Camellia sinensis is one of the most important economic trees in the world. Anthracnose caused by Colletotrichum spp. can jeopardize the leaf tissue of tea tree and seriously affect the yield of tea. It is the most economical and effective measure to control the tea tree anthracnose by using the resistance resources of the tea tree itself to cultivate new disease resistant varieties. The species and geographical distribution of the bacteria were not clear. At the same time, the role of secondary metabolites of tea tree was not clear. This paper systematically studied the biological diversity of the anthrax of Chinese tea tree, and took tea 108 (ZC108) and the susceptible variety Longjing 43 (LJ43) as the test material, and carried out the relationship between the anthrax and the tea tree. The main results are as follows: 1. 106 strains of anthrax were obtained from the leaves of tea varieties in the main tea producing areas of 15 provinces (municipalities and autonomous regions) in China. Based on the polygenic phylogenetic analysis (ACT, CAL, CHS-1, GAPDH, GS and ITS) and morphological analysis, these strains were found to belong to C.acutatum, C.boninense, C.cliviae, C.g respectively. Loeosporioides and C.truncatum are 11 species of 5 species, including 6 known species (C.camelliae, C.cliviae, C.fioriniae, C.fructicola, C.karstii and C.siamense), 3 new species (C.aenigma, C.endophytica and C.truncatum), 1 new species (C.wuxiense) and 1 undetermined species. The most species (7/11), including C.camelliae and C.fructicola, is the disease species of tea tree in China. At the same time, this study further validates the method of using ApMat and GS gene to quickly identify the interrelationship between C.gloeosporioides complex. The results show that this method does not completely distinguish between C.siamense and individual species, but it is still fast and effective. The results of pathogenicity test showed that C.camelliae, C.aenigma, C.endophytica, C.fructicola and C.truncatum could cause the pathogenesis of LJ43 leaves, while the other 6 species (C.cliviae, C.fioriniae, C.karstii, Colletotrichum sp., C.siamense and C.fructicola) were inoculated to observe anthrax The results of the germination of C.fructicola on different resistant varieties showed that after inoculation of 12 h, the C.fructicola of anthrax had begun to germinate on two varieties and formed attachments, but the number of spore germination and the number of attachments on the ZC108 leaves were obviously less than LJ43; after inoculation of 96h, the growth of anthrax C.fructicola in the resistant varieties was grown. The effects of H2O2 and anaphylactic necrosis (HR) on the interaction of different resistant varieties and anthrax C.fructicola were observed by two biphenyl diphenyl amine and trypan blue staining. The results showed that after inoculation of anthrax, the leaves of two varieties had H2O2 accumulation and HR reaction in varying degrees, but the resistant varieties were earlier than the susceptible varieties by 24h. The response degree was stronger than that of susceptible varieties, indicating that active oxygen and HR play an important role in the anti anthracnose process of tea tree,.4. inoculated ZC108 and LJ43 with anthrax C.fructicola, the total amount of polyphenols in leaves, catechin and caffeine content and their related synthetic genes. The results showed that (-) -EGCG, (+) the content of -C and caffeine and the expression of the key genes were induced by the induction of anthrax C.fructicola; indoor bacteriostasis test results showed that caffeine was better than tea polyphenols and catechin. The results of electron microscopy and physiological activity showed that caffeine could break the cell wall and protoplasm membrane of mycelium; and caffeine to tea tree caffeine. The analysis of the key gene TCS1 and SAMS promoter found that the promoter region contains several cis acting elements related to the resistance of plants. In summary, (+) -EGCG, (+) -C and caffeine play an important role in the resistance process of tea trees,.5. with ZC108 and LJ43 leaves as materials, and 0,24 after inoculation of anthrax C.fructicola. The results showed that each sample was sequenced in the transcriptional group. The results showed that each sample was sequenced to obtain at least 11.75 G clean base; the Trinity method was used to separate 2 varieties, 59336 unigenes and 35310 unigenes were obtained by LJ43, and all unigenes sequences were annotated by functional information after comparison with database AtNoPDB. 8 a total of 35935 unigenes were annotated and 35310 unigenes were annotated by LJ43; 19718 differentially expressed genes were screened by RSEM, and ZC108 obtained a total of 15848 differentially expressed genes, and 15848 of LJ43 were obtained. The differential expression genes of the differentially expressed genes were found to be enriched in the function related to disease resistance by the differential expression genes of the two varieties, but ZC108 through each other. The number of genes enriched in the road was obviously more than that of LJ43. on the KEGG enrichment analysis of differentially expressed genes. The differential expression genes of ZC108 were mainly enriched in plant hormone synthesis and caffeine metabolism pathway, while LJ43 was mainly enriched in the ribosome pathway. The above results showed that the synthesis and metabolism of plant hormones and caffeine were related to the anti anthrax of tea tree.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.711

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