【作者】 朱天辉；

【导师】 叶华智；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 由枯斑拟盘多毛孢菌(Pestalotiopsis funera Desm.)引起的松赤枯病是松树幼龄林上常见的病害，分布广、危害严重。该病在四川自1974年开始发生危害，到1980流行以来，已成为主要针叶林木病害，居林木病害首位。本文首次对枯斑拟盘多毛孢菌致病毒素的离体产生条件、毒素基本特性、专化性特点、毒素对寄主细胞质膜的伤害机制和对不同抗性松属植物细胞超微结构的影响及毒素活性成份分离纯化技术及化学结构进行了较系统的研究，以期了解枯斑拟盘多毛孢菌毒素的致病机理、不同松属植物的抗性反应及毒素活性物质的化学成份，从而为松属植物抗赤枯病育种和科学利用抗赤枯病树种提供可靠的理论基础。 本研究从四川松属植物分布区8个松属树种上采集松赤枯病样本，经单孢分离获得21个枯斑拟盘多毛孢菌菌株，所产毒素的致病性具明显的多样性。用马尾松、华山松、油松、云南松、湿地松、火炬松、辐射松、黑松一月龄左右的切根苗作生测材料，筛选出较强毒力的松赤枯病菌菌株(PF-1)作毒素产毒条件的研究，结果表明：枯斑拟盘多毛孢菌在七种参试培养基中，以PD培养基产毒最好，在培养基中加入敏感寄主马尾松松针浸出汁不能对毒素的产生和活性起促进作用。枯斑拟盘多毛孢菌在18-22℃、pH6.5-7.0、黑暗、静止状态下产毒最佳(15-27d)，这些条件与其菌丝生长、产孢有一定差异，表明枯斑拟盘多毛孢菌的生长与产毒条件不完全一致。 枯斑拟盘多毛孢菌毒素粗提液的基本特性研究显示：(1)毒素粗提液在80℃以下的温度有较高的稳定性，在高温(90-100℃)条件下，有失活现象发生；酸碱度(pH4-9)对其致病活性没有较为明显的影响。(2)该毒素不溶于乙酸乙酯等非极性溶剂，易溶于乙醇、甲醇等极性较大的溶剂。在氯仿与甲醇按不同比例提取时，随着甲醇比例的提高，提取液的致病活性也不断增强，据此可以推测枯斑盘多毛孢菌毒素可能为一类极性较大的物质(非蛋白类)，活性碳能对其吸附，并能用甲醇将其从培养液中较好地提取出来，这些特性的揭示为深入研究毒素提供了可靠的生化依据。 选用12种不同科属木本植物和15种不同科属杂草对毒素专化性研究表明，松属植物中，马尾松、油松、云南松对毒素最敏感、湿地松、火炬松次之，华山松、辐射松、黑松有较强的抗毒素能力，而不同科属的木本植物柳杉、杉木、南洋杉、兰桉对毒素也有不同程度的反应，说明该毒素为非专化性毒素。同时，不同生测材料试验证实松幼嫩的针叶对毒素的反应快而敏感，且能产生典型症状，是一种理想的生测材料。生测方法研究显示，与浸渍法、涂抹法相比，针刺法不仅反应快，而且用量少，用针刺法处理待测针叶，由于粗提液易为植物材料所吸收，在生测过程中不易长出霉菌，使生测结果更为直观和准确。本研究还表明枯斑盘多毛抱菌毒素能使十五种杂草中的九种产生不同程度的伤害，一方面反应出这种非寄主专化性毒素在除草上有一定选择性，另一方面，也表现出广谱的除草能力，这对于开发新型生物除草剂有一定的参考价值。 毒素对不同松属植物细胞膜的伤害通过离子渗漏(电导率换算成膜伤害指数)和丙二醛(州DA)含量变化评估:(l)马尾松(29.5/24h)、油松(28.5/2 4h)、云南松(25.7/24h)细胞膜伤害指数最大，湿地松(9 .0/2 4h)、火炬松(8 .0/24h)次之，华山松(3.9/2 4h)、辐射松(4.6/2 4h)、黑松(4.3/2 4h)最小，这种差异在一定程度上反映了不同松属植物在抗病性上的差异，同时研究还发现，随着毒素浓度的降低，寄主膜伤害指数(电导率)显著下降，感病树种(马尾松、油松、云南松)在24h时膜伤害指数达最大值，而抗病树种(华山松、辐射松、黑松)的膜伤害指数达最大值的时间有所推迟(48h)。 (2)不同松属植物在毒素处理后，针叶中的MDA含量变化趋势有显著差异，抗病树种针叶中MDA含量变化情况不如感病树种(马尾松、油松、云南松)明显，从而说明抗病树种(华山松、黑松、辐射松、湿地松、火炬松)细胞膜脂过氧化较低，对毒素的忍耐力较强，这实际上是一种抗病性在细胞膜的反应。 毒素对不同抗病性松属植物愈伤组织破坏的扫描电镜试验表明，正常愈伤组织表面结构丰满、圆滑，而毒素可造成松属植物愈伤组织表面结构的破坏，细胞皱缩、空瘪，这种破坏作用，随处理时间的延长而增大;并且，马尾松对毒素较华山松更为敏感，马尾松愈伤组织表面的毁坏程度比华山松大得多。另一方面，透射电镜显示，感病树种马尾松针叶经毒素处理后，细胞壁肿胀，细胞壁多处部分消解，细胞质膜断裂，散落在细胞中，细胞发生严重的质壁分离，叶绿体膜破坏、片层无序化，或片层模糊，线粒体脊模糊、无序化，脊消解;但抗病树种华山松针叶细胞结构破坏相对较小，常在同一视野中有的细胞受害而有的细胞基本正常，说明华山松对枯斑拟盘多毛菌毒素有一定的抗性。 毒素分离纯化及化学结构研究表明:(l)通过对薄层层析展开剂及柱层析填料的筛选，得出正丁醇:甲醇:水(60:巧:30)为最佳展开剂，硅胶H60型为最佳填料。(2)以生测为评判指标，用硅胶H60型柱反复柱层析，用正丁醇:甲醇:水(60:15:30)和不更多还原

【Abstract】 The pine needle blight caused by Pestalotiopsis funerea, a common and serious disease in young pine forest, has been the most important needle-leaf disease in Sichuan forest since the disease occurred in 1974 and became epidemic in 1980. The toxin production condition of P. funerea, the basic nature and host-specific characteristic of the toxin, the pathogenic mechanism of the toxin to host cell membrane, the effect of the toxin on the ultra-structure of cell in various resistant pines, and the purification and chemical structure analysis of the pathogenic composition were systematically studied hi this paper for the first time. These studies may result in understanding the pathogenic mechanism of the toxin, the resistant reaction of various pines to the toxin and the pathogenic composition of the toxin, and further a theory basis for the resistance breeding of pine to needle blight and scientific utilization of the resistant pines are expected to obtain.21 strain of P. funerea, toxin of which had obvious diversity in pathogenicity, were isolated by single spore method from 8 pines distributed in Sichuan. Pf-1 , stronger virulence, among 21 strain, was screened out to explore toxin production condition of P. funerea, with the cut-root-seedling of Pinus massoniana f,armandi, P.tabulaeformis, Ryunnanensis, P.elliottii, P.taeda,P.radiata and P.thunbergii as bio-determination materials. The fungus could reach the maximum toxin production in PD medium out of 7 tested culture liquid media, but the addition of pine needle extract to PD could not promote the toxin production and pathogenicity of this fungus. The fittest toxin production conditions of p. funerea were 18-22C , pH 6.5-7.0,dark,stilling culture for 15-27d, and there were a little difference between toxin production and hyphae growth, spore production conditions, that revealed that toxin production conditions of P. funerea were not completely in accord with those of growth of the fungus.Studies of the toxin crude extract traits showed: (1) The toxin crude extract was stronger resistant to less than 80C but the pathogenic composition of toxin crude extract was decomposed in high temperature condition(90-100C) , and pH 4-9 did not significantly affect the virulence of the toxin crude extract. (2) The toxin was not dissolved in non-polar solvent, such as ethyl acetate, but was dissolved in the higher-polar solvent, such as alcohol,methanol . With the solvents of various proportions of chloroform to methanol as extract solvents, the higher the proportion of methanol was, the stronger the pathogenicity of toxin extract was, therefore, it should be presumed that the toxin produced by P. funerea could be a higher-polar substance. The toxin, which could be absorbed by activated charcoal and be fully extracted by methanol from its culture liquid, was not a kind of protein material. All the characteristics of toxin provided biochemical basis for thorough study of toxin.12 arbor plants and 15 weeds from various families or genus were selected to study specification of toxin from P. funerea to host, the result showed that in pines, P. massoniana , P.tabulaeformis and P.yunnanensis were the most sensible to the toxin, P.elliottii , P.taeda for taking second place, and P.armandi, P.radiata and P.thunbergii were stronger resistant to the toxin, meanwhile, the various sensibilities of Cryptomeria fortunei, Cunningghamia lanceolata, Araucaria cunninghammi and Eucalyptus globules to the toxin were proved, these studies implied that the toxin belonged to a non-host specification toxin(NHST). On the other hand, the different bio-determination materials suggested that the immature pine needle, which was quick and sensitive to toxin and produced obvious infected symptom, was the ideal material for bio-determination. Compared with the methods of soaking and applying, acupuncture was the best method for bio-determination that had the features of quick reaction to toxin and low consumption of toxin. The fact that mold did not grow on the pine needles dealt with acupunctu更多还原