發(fā)布時(shí)間：2018-04-21 11:08

  本文選題：茶樹 + 炭疽病　； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：茶樹(Camellia sinensis)是世界重要的經(jīng)濟(jì)林木之一。由炭疽菌(Colletotrichum spp.)引起的炭疽病可以危害茶樹葉部組織,嚴(yán)重影響茶葉產(chǎn)量。利用茶樹自身抗病資源,培育抗病新品種是控制茶樹炭疽病最經(jīng)濟(jì)、有效的措施。目前,關(guān)于我國茶樹炭疽菌種類及其地理分布尚不清晰。同時(shí),茶樹次級代謝物對抗炭疽病的作用也不明確。本文系統(tǒng)研究了我國茶樹炭疽菌的生物多樣性,并以茶樹抗病品種中茶108(ZC108)和感病品種龍井43(LJ43)作為試驗(yàn)材料,對炭疽菌-茶樹互作關(guān)系進(jìn)行了較為深入的研究。主要結(jié)果如下:1.從我國15個(gè)省(市、自治區(qū))的茶葉主產(chǎn)區(qū)多個(gè)茶樹品種病葉上共分離獲得106株炭疽菌,基于多基因系統(tǒng)發(fā)育學(xué)(ACT、CAL、CHS-1、GAPDH、GS和ITS)和形態(tài)學(xué)分析,發(fā)現(xiàn)這些菌株分別歸屬于C.acutatum、C.boninense、C.cliviae、C.gloeosporioides和C.truncatum共5個(gè)復(fù)合種中的11個(gè)種,包括6個(gè)已知種(C.camelliae、C.cliviae、C.fioriniae、C.fructicola、C.karstii和C.siamense),3個(gè)新記錄種(C.aenigma、C.endophytica和C.truncatum),1個(gè)新種(C.wuxiense)和1個(gè)未確定種(Colletotrichum sp.)。C.gloeosporioides復(fù)合種的種類最多(7/11),其中C.camelliae和C.fructicola為我國茶樹優(yōu)勢致病種。同時(shí),本研究進(jìn)一步驗(yàn)證了利用ApMat和GS基因快速識別C.gloeosporioides復(fù)合種種間關(guān)系的方法,結(jié)果表明此方法并不能將C.siamense和個(gè)別種完全區(qū)分,但其依然是快速而有效判斷炭疽菌種級關(guān)系的重要手段。致病性測試結(jié)果表明,C.camelliae、C.aenigma、C.endophytica、C.fructicola和C.truncatum可使LJ43葉片致病,而其他6種(C.cliviae、C.fioriniae、C.karstii、Colletotrichum sp.、C.siamense和C.wuxiense)接種后未發(fā)病。2.利用組織透明法觀察炭疽菌C.fructicola在不同抗性品種上的萌發(fā)侵染情況。結(jié)果表明,接種12 h后,炭疽菌C.fructicola在兩個(gè)品種上均已開始萌發(fā),并形成附著胞,但在ZC108葉片上的孢子萌發(fā)數(shù)量和附著胞數(shù)量明顯少于LJ43;接種96h后,炭疽菌C.fructicola在抗病品種上的生長發(fā)育明顯延遲。3.利用二聯(lián)基聯(lián)苯胺和臺盼藍(lán)染色法,觀察不同抗性品種與炭疽菌C.fructicola互作中H2O2和過敏性壞死(HR)的作用。結(jié)果表明,在接種炭疽菌后,兩個(gè)品種葉片均出現(xiàn)不同程度的H2O2積累和HR反應(yīng),但抗病品種比感病品種提前24h響應(yīng),并且抗病品種葉片的反應(yīng)程度比感病品種強(qiáng),表明活性氧和HR在茶樹抗炭疽病過程中發(fā)揮著重要作用。4.分別對ZC108和LJ43接種炭疽菌C.fructicola,測定葉片多酚總量、兒茶素類和咖啡堿含量及其相關(guān)合成基因的變化。結(jié)果表明,(-)-EGCG,(+)-C和咖啡堿含量及其關(guān)鍵基因的表達(dá)受到炭疽菌C.fructicola誘導(dǎo)升高;室內(nèi)抑菌試驗(yàn)結(jié)果表明,咖啡堿的抑菌效果要優(yōu)于茶多酚和兒茶素;電鏡觀察結(jié)果和生理活性測定結(jié)果表明,咖啡堿能破壞菌絲的細(xì)胞壁和原生質(zhì)膜;對茶樹咖啡堿合成關(guān)鍵基因TCS1和SAMS啟動子序列進(jìn)行分析發(fā)現(xiàn),啟動子區(qū)域含有多個(gè)與植物抗逆性相關(guān)的順式作用元件。綜上所述,(-)-EGCG、(+)-C和咖啡堿在茶樹抗病過程中發(fā)揮了重要作用。5.分別以ZC108和LJ43葉片為材料,對接種炭疽菌C.fructicola后0、24和72h的葉片樣品進(jìn)行轉(zhuǎn)錄組測序。結(jié)果表明,每個(gè)樣品測序均獲得至少11.75 G clean base;利用Trinity法對2個(gè)品種進(jìn)行獨(dú)立組裝,ZC108共獲得59,336條unigenes,LJ43共獲得35,310條unigenes;將所有unigenes序列與數(shù)據(jù)庫AtNoPDB比對后進(jìn)行功能信息注釋,ZC108共注釋35,935條unigenes,LJ43共注釋35,310條unigenes;采用RSEM法篩選差異表達(dá)基因,ZC108共獲得19,718條差異表達(dá)基因,LJ43獲得15,848條;對差異表達(dá)基因進(jìn)行GO生物功能分析發(fā)現(xiàn),兩個(gè)品種的差異表達(dá)基因富集到與抗病有關(guān)的功能中,但ZC108各通路富集的基因數(shù)明顯多于LJ43。對差異表達(dá)基因進(jìn)行KEGG富集分析發(fā)現(xiàn),ZC108的差異表達(dá)基因主要富集在植物激素合成和咖啡堿代謝通路中,而LJ43主要富集在核糖體通路。以上結(jié)果表明植物激素和咖啡堿合成代謝與茶樹抗炭疽病有關(guān)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.711

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 賴建東;魏日鳳;連玲麗;劉偉;葉乃興;楊江帆;陳丹;楊國一;;炭疽病病原菌侵染對茶樹葉片生理特性的影響[J];福建農(nóng)林大學(xué)學(xué)報(bào)(自然科學(xué)版);2016年02期

2 凌光漢;陳小媛;;新昌縣茶樹炭疽病的發(fā)生與防治對策[J];中國茶葉;2015年12期

3 張秀娟;;紹興南部茶園害蟲發(fā)生特點(diǎn)及防治技術(shù)[J];現(xiàn)代農(nóng)業(yè)科技;2015年02期

4 李曉麗;羅榴彬;胡小倩;樓兵干;何勇;;基于共聚焦顯微拉曼光譜揭示炭疽病侵染下茶葉細(xì)胞壁變化的研究[J];光譜學(xué)與光譜分析;2014年06期

5 林德鋒;吳順章;黃大龍;張金文;;漳州鐵觀音茶炭疽病發(fā)生規(guī)律及化學(xué)防治試驗(yàn)[J];浙江農(nóng)業(yè)科學(xué);2012年08期

6 張亮;袁爭;朱蔚;高旭暉;;4種植物提取物對茶炭疽病菌的體外抑制作用[J];植物保護(hù);2012年04期

7 劉霞;江健;詹金碧;周開云;陳卓;;湄潭縣危害茶樹的主要病蟲害調(diào)查[J];貴州農(nóng)業(yè)科學(xué);2011年09期

8 陳百文;劉偉;趙恬歡;葉乃興;楊江帆;魏日鳳;;對茶樹炭疽病菌具拮抗作用的茶樹內(nèi)生細(xì)菌的篩選[J];福建農(nóng)林大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年04期

9 張華艷;戚麗;張正竹;;咖啡堿對茶樹主要葉部病原真菌的抑制作用[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);2010年02期

10 羅勇;吳江;陳健;;茶樹主要病害癥狀識別與防治[J];植物醫(yī)生;2010年01期

相關(guān)博士學(xué)位論文 前1條

1 岳川;茶樹糖類相關(guān)基因的挖掘及其在茶樹冷馴化中的表達(dá)研究[D];中國農(nóng)業(yè)科學(xué)院;2015年

相關(guān)碩士學(xué)位論文 前2條

1 白靜科;果生炭疽菌Colletotrichum fructicola與蘋果不同抗性品種互作研究[D];西北農(nóng)林科技大學(xué);2016年

2 劉威;茶樹炭疽病的病原鑒定及其遺傳多樣性分析[D];福建農(nóng)林大學(xué);2013年

，

本文編號：1782181