【作者】 房保海；

【导师】 张广民；

【作者基本信息】 山东农业大学 ， 植物病理学， 2003， 硕士

【摘要】 烟草低头黑病(Colletotrichum capsici（Sdy）Bulter & Bisby f. nicotianae G.M.Zhang & G.Z.Jiang)是危害烟草的主要病害之一，造成大量减产。1995年，张广民首次证明烟草低头黑病菌能产生对烟草有致萎作用的毒素类物质，所以对烟草低头黑病菌毒素及其作用特性的研究对于揭示烟草低头黑病菌与烟草的互作机制有重要意义。本文分别对烟草低头黑病菌产毒菌株、产毒条件、烟草低头黑病菌毒素的一些基本性质、TLC分析初探、毒素对烟草膜透性的影响以及对烟草植株体内丙二醛（MDA）含量和某些防御酶的影响进行了研究。所得研究结果如下:1. 烟草低头黑病菌不同菌株皆能产生使烟苗快速萎蔫的毒素类物质，排除了由于溶液渗透势导致萎蔫的可能性，以菌株C-Ⅲ-1、C-Ⅴ-1和C-Ⅲ-5产毒较强，C-Ⅲ-1最强。实验表明低头黑病菌产生毒素的最佳条件为：pH=6和25℃～30℃条件下连续黑暗振荡培养13d，适宜培养基为PDB、烟草煎汁和麦芽糖液体培养基。利用活体植株浸渍法和离体叶片针刺接种法对10个科的25种植物测定了烟草低头黑病菌毒素的致毒范围，发现烟草低头黑病菌毒素能够侵染茄科、豆科、藜科、葫芦科、十字花科和菊科6个科的12种植物，其中以茄科的普通烟、心叶烟最为敏感。2. 对烟草低头黑病菌毒素的基本性质研究表明：烟草低头黑病菌毒素在70℃处理20min对致毒活性没有显著的影响、在pH4-pH9范围内具有酸碱稳定性；紫外光对毒素的致毒活性具有一定的消弱作用。经丙酮沉淀处理、透析膜和木瓜蛋白酶处理后的生测结果表明，低头黑病菌毒素属于低分子量（Mr）的非蛋白类物质。烟草低头黑病菌毒素能够溶于乙酸乙酯、三氯甲烷和正丁醇，不溶于苯；低头黑病菌毒素的有效成分在酸性环境下更易溶于乙酸乙酯。紫外光谱扫描表明烟草低头黑病菌毒素在波长248、252、264、272 、284nm 都有吸收峰。对烟草低头黑病菌粗毒素进行硅胶G薄层层析分离，获得3个活性成分：组分Ⅲ（Rf=0.41）、Ⅳ（Rf=0.47）和Ⅶ（Rf=0.67）。3. 烟草低头黑病菌毒素能增加烟草叶片和根部细胞的透性，造成电解质外渗，且随着处理时间的延长和毒素浓度的提高这种作用更明显；感病品种对毒素更敏感。毒素接种后期叶绿素明显降低。烟草低头黑病菌毒<WP=7>素处理的叶片组织结构严重受损，细胞壁分解成碎片，很多细胞表现为膨胀。表明烟草低头黑病菌毒素对烟草的作用位点可能位于细胞膜和叶绿体上。4. 烟草低头黑病菌毒素对烟草植株内某些防御酶的影响：用烟草低头黑病病菌毒素处理后，抗病品种Burley21的SOD活性初期有一缓慢下降阶段（2h~12h），感病品种NC82的SOD活性明显低于对照，但表现为上升趋势；抗病品种Burley21的SOD活性于36h达到高峰，而后下降，但抗病品种Burley21的SOD活性一直高于感病品种NC82。Burley21和NC82毒素处理后，POD活性表现为上升，48h达到高峰；Burley21的POD的活性一直维持较高水平，而NC82的POD活性则迅速下降至对照水平。抗感品种PPO活性处理早期均升高，后期下降；但抗病品种PPO活性一直高于感病品种和对照。抗病品种阴极端碱性POD谱带a（Rf=0.29）、b （Rf=0.35）和阳极端酸性POD谱带强度都高于感病品种，而且比感病品种NC82多出c （Rf=0.46）、d（Rf=0.54）两条谱带。5. 烟草低头黑病菌毒素对烟草MDA含量的影响: 膜脂过氧化产物MDA在毒素处理早期含量升高（12h），后有所下降，但后期MDA含量又重新上升，抗病品种Burley21的MDA增减比率波动比感病品种NC82小。更多还原

【Abstract】 Tobacco black death disease(Colletotrichum capsici（Sdy）Bulter & Bisby f. nicotianae G.M.Zhang & G.Z.Jiang) is an important tobacco disease , which made yield decline abruptly. In 1995, Zhang Guangmin firstly certified that Colletotrichum capsici f. nicotianae could produce toxin-like substance causing tobacco wilt. So further studies on Colletotrichum capsici f. nicotianae toxin are of great significance to reveal the interaction between Colletotrichum capsici f. nicotianae and tobacco. This dissertation made studies on screening toxin-producing strains, basic properties of Colletotrichum capsici f. nicotianae toxin, preliminary TLC analysis about rough toxin, effects of the toxin on membrane permeability, MDA amount and some defensive enzymes respectively. The results were as follows: 1. Colletotrichum capsici f. nicotianae strains all can produce toxin that can make tobacco wilt, and the toxicity is not from osmotic potential. Strain C-Ⅲ-1, C-Ⅴ-1 and C-Ⅲ-5 had stronger capability of producing toxin than the others, and C-Ⅲ-1 is the strongest one. Results showed that the optimum condition of producing toxin was: under the condition of pH6, 25℃～30℃, and 13 days of continuous oscillation culture; PDB, tobacco leaf frying juice and maltose liquid cultural medium were the three suitable cultural media. With plant pickling in vivo method and excised leaf acupuncture inoculation method, we found that among 10 families and 25 species of plants Colletotrichum capsici f. nicotianae toxin could infect Solanaceae，Leguminosae，Chenopodiaceae，Cucurbitaceae，Cruciferae，6 families and<WP=9>12 species of plants，in which Nicotianae tabacum， and N. glutinosa were most susceptible.2. Studies on Colletotrichum capsici f. nicotianae toxin showed that: the toxin was thermostable after being treated for 20min in 70℃ and acid-basically stable in the range of pH4-pH9; ultraviolet radiation could tenuate its toxicity. After being treated by alcohol precipitation， dialysis membrane and papain，the bioassay results showed that Colletotrichum capsici f. nicotianae toxin belonged to small molecular weight and non-protein. Colletotrichum capsici f. nicotianae toxin could dissolve in organic solvent ethyl acetate， chloroform，n-butyl alcohol，and sodium bicarbonate saturated solution; and could not in benzene; the active constituent could dissolve in ethyl acetate more easily in acid condition.Ultraviolet spectral scanning analysis showed that the toxin had absorption peak at wavelength 248,252,264,272 and 284nm. 3 active constituents were obtained by TLC segragation:Ⅲ(Rf=0.41),Ⅳ(Rf=0.47) and Ⅶ(Rf=0.67).3. Colletotrichum capsici f. nicotianae toxin could increase the cell permeability of tobacco leaf and root, which caused electrolyte leakage，and this kind of action is more violent with the prolongation of time and the increasing of toxin concentration; Susceptible tobacco is more sensitive to the toxin. The toxin could decrease the content of tobacco leaf chlorophyll. There are great changes in tobacco leaf microstructure after being treated by the toxin; cell wall was broken into fragment and many cells swelled. That showed that Colletotrichum capsici f. nicotianae toxin action position might be on the cell membrane and chloroplast.4. Resistant tobacco Burley21 and susceptible tobacco NC82 were inoculated with rough toxin respectively，and the changes of the defensive enzyme activity were detected after inoculation. For SOD, the activity of resistant tobacco descended firstly in early time(2h-12h)，but that of susceptible tobacco ascended and was lower than the control. SOD activity of resistant tobacco reached the peak at 36h and subsequently descended，but the<WP=10>activity were higher than that of the susceptible at all the inoculation time. For POD, the activity of resistant tobacco and susceptible tobacco both ascended after inoculation and reached the peak at 48h. Subsequently POD activity of resistant tobacco could keep high level，but that of susceptible tobacco came down to the control lev更多还原