稻曲病菌毒素活性及致病性细胞化学研究
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摘要
稻曲病是世界性水稻真菌病害，致病菌属于子囊菌纲球壳菌目麦角菌科绿核菌属(Ustilaginodea virens(cooke)Tak)。稻曲病过去仅在中晚稻田零星发生，所以一直不被人们重视，被世界各地作为次要病害防治。但从上世纪80年代以后，随着水稻品种的改变及稻田肥力的增加，稻曲病的发生日益严重，在一些地区已成为影响水稻产量和质量的重要因素。
稻曲病不仅危害水稻穗部形成稻曲球，造成减产并污染水稻，影响水稻品质，更重要的含有对人畜有害的毒素。目前，国内外对毒素的研究，多侧重于对人畜毒性的研究，而对毒素在病原菌致病过程中的作用和毒素对水稻毒性的研究较少，病理学研究较为薄弱。本课题主要对稻曲病菌毒素进行系统的研究，对稻曲病菌毒素的生物学活性，毒素的分泌行为及其毒素在致病过程中的作用进行深入的探讨，为探索有效防治稻曲病的策略和抗病育种提供科学依据，主要有以下几个方面的结果：
1．病原菌分离、粗毒素的提纯与活性测定：稻曲病菌在固体培养基上生长缓慢，用PD培养液或PS培养液均可获得大量的培养滤液，但以PD培养的量较大。生物学活性测定结果表明，用培养20天左右的培养滤液提取毒素为好。用100％甲醇提取粗毒素比较好，而用70％饱和(NH_4)_2SO_4溶液沉淀法，因为涉及到生物学活性测定，而其中的NH_4~+，SO_1~(2-)离子不能完全有效去除，影响生物学活性测定的准确性，所以不适合用于稻曲病毒素的分离。生长抑制实验表明，稻曲病菌毒素对胚根的抑制作用明显高于对胚芽抑制作用，可能是由于胚根直接接触毒素的结果，所以采用胚根生长抑制率作为稻曲病菌毒素的生物活性测定较为合适。
2．稻曲病菌毒素Ustiloxin A多克隆抗体的制备与免疫胶体会检测：制备了稻曲病菌毒素Ustiloxin A的多克隆抗体，ELISA检测表明用两种偶联剂偶联所制备的抗体效价分别为1∶20000和1∶6000，并在细胞水平上检测了毒素的分泌行为，肯定了抗体的特异性。通过免疫细胞化学标记，我们分析了稻曲病菌分泌毒素的行为过程，并且分析了田间稻曲球中的毒素的分泌情况。
3．毒素对植物细胞分裂的抑制作用分析：用大蒜根尖做抑制试验，发现用稻曲病菌培养滤液处理过的大蒜根尖呈分裂末期的细胞数目明显多于相同条件
浙江大学硕士学位论文
下对照的数目，抑制率为69%，且细胞明显比对照的细胞大，说明毒素抑制根尖
细胞的有丝分裂，但不能抑制细胞的伸长。
False smut is a worldwide rice disease caused by Ustilaginodea virens (cooke) Tak. It was all along ignored before 1980 because of its occasionally happening. Since 1980s', however, with the change of rice cultivar and adding of more manure into the paddyfield, false smut has become worse and worse as an important factor to influence greatly on the yield and quality of rice in some places.
The false smut balls grow parasitically on panicle of rice plant, results in less yield, polluted rice and worse quality, and most important, produces toxins poisoning to human beings and domestic animals. By far, more study about ustiloxins is focused on its toxicity to human beings and domestic animals and less on the role of ustiloxins in the form of the disease, the toxicity to rice plant itself and its pathology. In order to seek for the effective means of prevention and cure of false smut and provide scientific evidences to the breeding of cultivar resisting to disease, we systematically studied the ustiloxins of false smut, including the biological activity of ustiloxins, the synthesis and secretion of ustiloxins and the role of ustiloxins in the form of the disease. The main results are as follows:
1. Culturing pathogen, isolating rough ustiloxins and mensurating biological activity: Ustilaginodea virens growed slowly in the solid culture, but well in PD or PS medium. The mensuration of biological activity showed that it was better to isolate ustiloxins using culture filtrate cultured for about 20 days. More appropriate way to isolate rough ustiloxins was 100% methyl alcohol rather than 70% (NH4)2 SO1, solution because it involved the incompletely removed ions, NH4+and SO42-, which disturbed the mensuration of biological activity. The experiment of growth restrain indicated that growth restrain ratio of radicle was appropriate to be used in the mensuration of biological activity because inhibition of the rough ustiloxins to the embryo was significantly weaker than to the radicle, possibly its directly touching the ustiloxins.
2. Preparing antiserum against Ustiloxin A and mensurating it with immuno-gold: The antiserum against Ustiloxin A, a main component of the toxins, was prepared by immunizing New Zealand rabbit. Elisa assay showed that the liters
of the two antiserums obtained were 1:20000 and 1:6000, respectively. The specificity of the antiserum was confirmed by immuno-gold labeling and the synthesis and secretion of Ustiloxin A was examined at the cell level. We also analyzed the processes of secretion of Ustiloxin A in culture medium and in false smut ball in the field by immuno-gold labeling.
3. Analysing ustiloxins inhibiting plant cell mitosis The inhibition experiment of growth of garlic radicle showed that there were more cells in the telophase stage in the garlic radicle dealed with culture filtrate of Ustilaginodea virens than those dealed with H2O. The cell volume of the former was bigger than that of the latter. So it
seemed that ustiloxins could inhibit the cell mitosis, but not in the cell elongation.
稻曲病不仅危害水稻穗部形成稻曲球，造成减产并污染水稻，影响水稻品质，更重要的含有对人畜有害的毒素。目前，国内外对毒素的研究，多侧重于对人畜毒性的研究，而对毒素在病原菌致病过程中的作用和毒素对水稻毒性的研究较少，病理学研究较为薄弱。本课题主要对稻曲病菌毒素进行系统的研究，对稻曲病菌毒素的生物学活性，毒素的分泌行为及其毒素在致病过程中的作用进行深入的探讨，为探索有效防治稻曲病的策略和抗病育种提供科学依据，主要有以下几个方面的结果：
1．病原菌分离、粗毒素的提纯与活性测定：稻曲病菌在固体培养基上生长缓慢，用PD培养液或PS培养液均可获得大量的培养滤液，但以PD培养的量较大。生物学活性测定结果表明，用培养20天左右的培养滤液提取毒素为好。用100％甲醇提取粗毒素比较好，而用70％饱和(NH_4)_2SO_4溶液沉淀法，因为涉及到生物学活性测定，而其中的NH_4~+，SO_1~(2-)离子不能完全有效去除，影响生物学活性测定的准确性，所以不适合用于稻曲病毒素的分离。生长抑制实验表明，稻曲病菌毒素对胚根的抑制作用明显高于对胚芽抑制作用，可能是由于胚根直接接触毒素的结果，所以采用胚根生长抑制率作为稻曲病菌毒素的生物活性测定较为合适。
2．稻曲病菌毒素Ustiloxin A多克隆抗体的制备与免疫胶体会检测：制备了稻曲病菌毒素Ustiloxin A的多克隆抗体，ELISA检测表明用两种偶联剂偶联所制备的抗体效价分别为1∶20000和1∶6000，并在细胞水平上检测了毒素的分泌行为，肯定了抗体的特异性。通过免疫细胞化学标记，我们分析了稻曲病菌分泌毒素的行为过程，并且分析了田间稻曲球中的毒素的分泌情况。
3．毒素对植物细胞分裂的抑制作用分析：用大蒜根尖做抑制试验，发现用稻曲病菌培养滤液处理过的大蒜根尖呈分裂末期的细胞数目明显多于相同条件
浙江大学硕士学位论文
下对照的数目，抑制率为69%，且细胞明显比对照的细胞大，说明毒素抑制根尖
细胞的有丝分裂，但不能抑制细胞的伸长。
False smut is a worldwide rice disease caused by Ustilaginodea virens (cooke) Tak. It was all along ignored before 1980 because of its occasionally happening. Since 1980s', however, with the change of rice cultivar and adding of more manure into the paddyfield, false smut has become worse and worse as an important factor to influence greatly on the yield and quality of rice in some places.
The false smut balls grow parasitically on panicle of rice plant, results in less yield, polluted rice and worse quality, and most important, produces toxins poisoning to human beings and domestic animals. By far, more study about ustiloxins is focused on its toxicity to human beings and domestic animals and less on the role of ustiloxins in the form of the disease, the toxicity to rice plant itself and its pathology. In order to seek for the effective means of prevention and cure of false smut and provide scientific evidences to the breeding of cultivar resisting to disease, we systematically studied the ustiloxins of false smut, including the biological activity of ustiloxins, the synthesis and secretion of ustiloxins and the role of ustiloxins in the form of the disease. The main results are as follows:
1. Culturing pathogen, isolating rough ustiloxins and mensurating biological activity: Ustilaginodea virens growed slowly in the solid culture, but well in PD or PS medium. The mensuration of biological activity showed that it was better to isolate ustiloxins using culture filtrate cultured for about 20 days. More appropriate way to isolate rough ustiloxins was 100% methyl alcohol rather than 70% (NH4)2 SO1, solution because it involved the incompletely removed ions, NH4+and SO42-, which disturbed the mensuration of biological activity. The experiment of growth restrain indicated that growth restrain ratio of radicle was appropriate to be used in the mensuration of biological activity because inhibition of the rough ustiloxins to the embryo was significantly weaker than to the radicle, possibly its directly touching the ustiloxins.
2. Preparing antiserum against Ustiloxin A and mensurating it with immuno-gold: The antiserum against Ustiloxin A, a main component of the toxins, was prepared by immunizing New Zealand rabbit. Elisa assay showed that the liters
of the two antiserums obtained were 1:20000 and 1:6000, respectively. The specificity of the antiserum was confirmed by immuno-gold labeling and the synthesis and secretion of Ustiloxin A was examined at the cell level. We also analyzed the processes of secretion of Ustiloxin A in culture medium and in false smut ball in the field by immuno-gold labeling.
3. Analysing ustiloxins inhibiting plant cell mitosis The inhibition experiment of growth of garlic radicle showed that there were more cells in the telophase stage in the garlic radicle dealed with culture filtrate of Ustilaginodea virens than those dealed with H2O. The cell volume of the former was bigger than that of the latter. So it
seemed that ustiloxins could inhibit the cell mitosis, but not in the cell elongation.
引文
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