水稻稻曲球及稻曲病菌厚垣孢子休眠特性研究
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摘要
水稻稻曲病(Rice False Smut)是由稻绿核菌[Ustilaginoidea virens (Cke)Tak]引起的水稻穗部真菌性病害。该病在世界各水稻产区均有发生,近年来呈现加重趋势,它不仅直接影响水稻的品质和产量,而且其产生的毒素对人和动物均有毒性。
本研究对不同颜色稻曲球采用进行石蜡切片观察其内部结构,结果表明黄色和黑色稻曲球均分为3层：厚垣孢子层、菌丝层、胚乳层,且胚乳层与菌丝层分界明显。黄色稻曲球颗粒花药结构完整,具有花粉囊及花粉粒,黑色稻曲球颗粒仅有部分花药外壁(主要为纤维素和木质素),其他部分已被病菌侵染。
对中稻和晚稻稻曲病菌的不同颜色厚垣孢子用扫描电子显微镜(SEM)和透射电子显微镜(TEM)进行表面和内部超微结构观察,结果表明：在SEM下,黄色和黑色厚垣孢子均为球形或近球形,黄色厚垣孢子直径为3-6μm,黑色厚垣孢子直径为4-6μm；黄色厚垣孢子表面具有疣状突起,突起高度为200-700nm,黑色厚垣孢子表面为锥形刺状突起,突起高度为400-1100nm,在TEM下,黄色和黑色厚垣孢子均由内外两层壁组成,内壁厚于外壁；黄色厚垣孢子内细胞核等细胞器明显可见,黑色厚垣孢子内能看到一大的脂质球,细胞质被挤压至脂质球外围。经稻曲病菌两种不同颜色厚垣孢子的表面和内部超微结构比较,其超微结构均有差异,由此表明稻曲病菌厚垣孢子转入休眠与其结构变化有关。
采用了4种机械破壁和两种酶(蜗牛酶和溶菌酶)对该病菌黄色和黑色厚垣孢子进行破壁,选取最佳破壁方案,结果表明液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,该法破壁测得的厚垣孢子壁内可溶性蛋白质含量也最高。得出液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。
采用碱提法提取不同颜色厚垣孢子壁内的蛋白质及多糖,分别用考马斯亮蓝G-250法测定蛋白质含量,硫酸蒽酮法测定多糖含量,结果表明：黄色厚垣孢子壁内含蛋白质9.698%,多糖73.867%；黑色厚垣孢子壁内蛋白质含量为14.187%,多糖含量为81.207%。
Rice false smut is a fungal disease caused by Ustilaginoidea virens (Cke)Tak, which harmed the panicles of rice. Rice false smut is a worldwide disease, and became more and more seriously in recently years, which not only influence greatly on the yield and quality of rice, but ustiloxins also cause poisoning to human and animals.
This study observed the structure of different color rice false smut ball by using paraffin section, and found that the rice false smut balls was divided into three layers: Chlamydospore mound;endosperm layer; subiculum, and there was no crossed interface between the subiculum and endosperm layer. In yellow rice false smut balls, an anther with complete pollen sac and pollen grain; in black one there was only part of the outer wall of anther (mainly contained cellulose and lignin), other parts has been infected. The surface and internal structural differences of yellow and black dormant chlamydospores of Ustilaginoidea virens on mid-and late-season rice were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM revealed that the spherical yellow and black chlamydospores had prominent spines, with diameters from 3 to 6μrn and 4 to 6μm, respectively. The spines were 200-700 nm and 400-1100 nm long in yellow and black chlamydospores, respectively. TEM revealed that the cell wall of both color chlamydospores were composed of two layers, the thick endosporium and thin exosporium, and the thickness of the former layer of the black chlamydospores was nearly two times more than that of the yellow ones. The organelles in yellow chlamydospores such as the nucleus were obviously visible, but invisible in black chlamydospores. The cytoplasm of black chlamydospores was occupied by a big lipid globule.
To explore the optimum method to wall-breaking of chlamydospores of the rice false smut fungus, Four wall-breaking methods and two enzymes (lysozyme and snailase) were been used. Results showed that Liquid nitrogen grinding plus ultrasonic crushing technique was the best one, its wall-breaking rates on yellow and black chlamydospores could all reached over 98%, and the soluble protein content also achieved the highest values. Therefore, liquid nitrogen grinding plus ultrasonic crushing method is an effective, economic, convenient wall-breaking method for U. virens in the laboratory.
Alkali extract was used to extract the cell wall protein and polysaccharides, and studied with Coomassie Brilliant Blue(CBB G-250)method and the method of anthrone, respectively. Result revealed that the cell wall of yellow chlamydospores with protein 9.698% and polysaccharide 73.867%, and cell wall of black chlamydospores with protein 14.187% and polysaccharide 81.207%.
本研究对不同颜色稻曲球采用进行石蜡切片观察其内部结构,结果表明黄色和黑色稻曲球均分为3层：厚垣孢子层、菌丝层、胚乳层,且胚乳层与菌丝层分界明显。黄色稻曲球颗粒花药结构完整,具有花粉囊及花粉粒,黑色稻曲球颗粒仅有部分花药外壁(主要为纤维素和木质素),其他部分已被病菌侵染。
对中稻和晚稻稻曲病菌的不同颜色厚垣孢子用扫描电子显微镜(SEM)和透射电子显微镜(TEM)进行表面和内部超微结构观察,结果表明：在SEM下,黄色和黑色厚垣孢子均为球形或近球形,黄色厚垣孢子直径为3-6μm,黑色厚垣孢子直径为4-6μm；黄色厚垣孢子表面具有疣状突起,突起高度为200-700nm,黑色厚垣孢子表面为锥形刺状突起,突起高度为400-1100nm,在TEM下,黄色和黑色厚垣孢子均由内外两层壁组成,内壁厚于外壁；黄色厚垣孢子内细胞核等细胞器明显可见,黑色厚垣孢子内能看到一大的脂质球,细胞质被挤压至脂质球外围。经稻曲病菌两种不同颜色厚垣孢子的表面和内部超微结构比较,其超微结构均有差异,由此表明稻曲病菌厚垣孢子转入休眠与其结构变化有关。
采用了4种机械破壁和两种酶(蜗牛酶和溶菌酶)对该病菌黄色和黑色厚垣孢子进行破壁,选取最佳破壁方案,结果表明液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,该法破壁测得的厚垣孢子壁内可溶性蛋白质含量也最高。得出液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。
采用碱提法提取不同颜色厚垣孢子壁内的蛋白质及多糖,分别用考马斯亮蓝G-250法测定蛋白质含量,硫酸蒽酮法测定多糖含量,结果表明：黄色厚垣孢子壁内含蛋白质9.698%,多糖73.867%；黑色厚垣孢子壁内蛋白质含量为14.187%,多糖含量为81.207%。
Rice false smut is a fungal disease caused by Ustilaginoidea virens (Cke)Tak, which harmed the panicles of rice. Rice false smut is a worldwide disease, and became more and more seriously in recently years, which not only influence greatly on the yield and quality of rice, but ustiloxins also cause poisoning to human and animals.
This study observed the structure of different color rice false smut ball by using paraffin section, and found that the rice false smut balls was divided into three layers: Chlamydospore mound;endosperm layer; subiculum, and there was no crossed interface between the subiculum and endosperm layer. In yellow rice false smut balls, an anther with complete pollen sac and pollen grain; in black one there was only part of the outer wall of anther (mainly contained cellulose and lignin), other parts has been infected. The surface and internal structural differences of yellow and black dormant chlamydospores of Ustilaginoidea virens on mid-and late-season rice were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM revealed that the spherical yellow and black chlamydospores had prominent spines, with diameters from 3 to 6μrn and 4 to 6μm, respectively. The spines were 200-700 nm and 400-1100 nm long in yellow and black chlamydospores, respectively. TEM revealed that the cell wall of both color chlamydospores were composed of two layers, the thick endosporium and thin exosporium, and the thickness of the former layer of the black chlamydospores was nearly two times more than that of the yellow ones. The organelles in yellow chlamydospores such as the nucleus were obviously visible, but invisible in black chlamydospores. The cytoplasm of black chlamydospores was occupied by a big lipid globule.
To explore the optimum method to wall-breaking of chlamydospores of the rice false smut fungus, Four wall-breaking methods and two enzymes (lysozyme and snailase) were been used. Results showed that Liquid nitrogen grinding plus ultrasonic crushing technique was the best one, its wall-breaking rates on yellow and black chlamydospores could all reached over 98%, and the soluble protein content also achieved the highest values. Therefore, liquid nitrogen grinding plus ultrasonic crushing method is an effective, economic, convenient wall-breaking method for U. virens in the laboratory.
Alkali extract was used to extract the cell wall protein and polysaccharides, and studied with Coomassie Brilliant Blue(CBB G-250)method and the method of anthrone, respectively. Result revealed that the cell wall of yellow chlamydospores with protein 9.698% and polysaccharide 73.867%, and cell wall of black chlamydospores with protein 14.187% and polysaccharide 81.207%.
引文
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