玉米南方锈病的分子检测与玉米抗病性机制的研究
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摘要
玉米南方锈病(Puccinia polysora Underw.)是近年来在我国发生明显呈上升趋势的病害,对我国玉米生产造成严重的威胁。为了研究玉米南方锈病流行规律以及抗病机制,本试验开展了病菌特异性检测的技术研究,并通过曲利苯兰透明染色法对南方锈菌侵染不同抗病性玉米自交系的组织病理学进行了研究,同时对玉米不同抗病性自交系在苗期进行接种后的生理生化指标进行了测定。找出了与玉米抗南方锈病相关的生理生化指标。本研究主要结果如下:
1利用真菌的ITS通用引物ITS1-F和ITS4-B对玉米南方锈菌DNA进行扩增,经过回收、克隆、测序,获得病菌DNA的一段905 bp的DNA片段。用软件primer 5设计了一对特异性引物用于对南方锈菌的分子检测。用此对引物对常见的几种玉米病原真菌与健康植株组织DNA进行扩增,结果只有以南方锈菌基因组DNA为模板的反应体系能扩增出一条356bp的特异性条带,其余基因组DNA模板均无扩增产物产生。表明此特异性引物可以用于玉米组织内南方锈菌的分子检测及病害的早期诊断。经灵敏度试验测定表明,该对引物可以检测到10 pg/uL的P. polysora基因组DNA。
2采用曲利苯兰透明染色法,以4个玉米自交系为材料,研究了玉米南方锈病菌在不同抗病性品种上侵染过程的组织学特征。结果表明,南方锈病菌侵入和定殖可以分为5个阶段:孢子萌发与芽管形成、附着胞形成、侵入细胞、吸器产生、菌丝在细胞间扩展。在不同抗病性的玉米材料上,病菌孢子萌发和芽管形成差别不明显,但侵入后的病菌在不同抗病性材料内的发育进程和发育程度具有显著差异。在抗病玉米材料,病菌初生菌丝、吸器母细胞、次生菌丝的形成时间推迟,吸器少,菌丝分枝少,菌丝生长缓慢。
3抗感自交系经过接种处理后,可溶性蛋白含量都有所下降,与抗病性之间无明显相关性。感病自交系体内可溶性糖含量始终高于抗病自交系。游离脯氨酸含量与苯丙氨酸解氨酶(PAL)酶活在抗病自交系体内增加幅度高于感病自交系。多酚氧化酶(PPO)酶活在抗感自交系体内变化规律与抗病性之间无明显的相关性。抗病自交系体内超氧化物歧化酶(SOD)与过氧化物酶(POD)酶活增幅高于感病自交系,且能长时间维持在较高水平。抗病自交系体内过氧化氢酶(CAT)酶活始终高于对照和感病自交系。因此在玉米苗期,其叶片内可溶性糖和游离脯氨酸含量、PAL、SOD、POD及CAT酶活可作为玉米抗南方锈病的生化指标。
Southern corn rust, caused by Puccinia polysora Underw., is becoming serious in recent years and is a potential threat for maize production in China. With the purpose of studying on the epidemic regularity and resistance mechanism of southern corn rust, specific detection of the pathogen was carried out, and the histopathological characters of P. polysora during infection and colonization in four maize inbred lines were investigated by trypan blue clearing staining technique. Meanwhile, the dynamic change of biochemical indicators was determined. The biochemical indicators associated with southern corn rust resistance were figured out. The main results of this study were as follows:
1. Southern corn rust DNA was amplified by universal primer, and the ITS sequences were obtained after gel extraction, cloning, and sequencing. The length ITS was 692 bp. Primers specific for P. polysora were designed using the primer-design software primer 5. The specific primers were used to amplify some fungal pathogens of maize and healthy tissues. A fragments 365 bp in length was amplified from the pathogen P. polysora only. The detection sensitivity was 10 pg/μL DNA.
2. The histopathological characters of P. polysora during infection and colonization in four maize inbred lines were investigated by trypan blue clearing staining technique. The process of pathogen invasion included five phases: spore germination and germ tube formation, appressorium formation, infection, formation of haustorium, and expansion of mycelium. The differences between spore germination and germ tube information were not very distinct. On the countary, when the pathogen invaded into the host cells of resistant maize lines, the growth and expansion degree of hyphae were distinguished. In the resistant lines, it was later to form primary hyphae, haustoriums and secondery hyphae, and less of haustoria in cells, fewer branches of hyphae and lower growth of hyphae than that in the susceptable lines.
3. After inoculation, the soluble protein contents in resistant and susceptible inbred lines were reduced, which were not associated with disease resisitance. The dissoluble sugar content was higher in the susceptible inbred lines than that in the resistant inbred lines. The resistant inbred lines showed higher increasing rate of the free proline content and the activity of PAL than the susceptible inbred lines. There was no certain correlation on resisitance of the activity of PPO in resistant and susceptible inbred lines. The resistant inbred lines showed higher increasing rate of the activity of SOD and POD. Furthermore, the activity of SOD and POD maintained high level throughout the study. The activity of CAT of the resistant inbred lines was always higher than the susceptible inbred lines and the control. Therefore, at the seedling stage of maize, the dissoluble sugar and the free proline contents and the activity of PAL, SOD, POD, and CAT in leaves can be used as the indictors for screening resistance to P. polysora in maize.
1利用真菌的ITS通用引物ITS1-F和ITS4-B对玉米南方锈菌DNA进行扩增,经过回收、克隆、测序,获得病菌DNA的一段905 bp的DNA片段。用软件primer 5设计了一对特异性引物用于对南方锈菌的分子检测。用此对引物对常见的几种玉米病原真菌与健康植株组织DNA进行扩增,结果只有以南方锈菌基因组DNA为模板的反应体系能扩增出一条356bp的特异性条带,其余基因组DNA模板均无扩增产物产生。表明此特异性引物可以用于玉米组织内南方锈菌的分子检测及病害的早期诊断。经灵敏度试验测定表明,该对引物可以检测到10 pg/uL的P. polysora基因组DNA。
2采用曲利苯兰透明染色法,以4个玉米自交系为材料,研究了玉米南方锈病菌在不同抗病性品种上侵染过程的组织学特征。结果表明,南方锈病菌侵入和定殖可以分为5个阶段:孢子萌发与芽管形成、附着胞形成、侵入细胞、吸器产生、菌丝在细胞间扩展。在不同抗病性的玉米材料上,病菌孢子萌发和芽管形成差别不明显,但侵入后的病菌在不同抗病性材料内的发育进程和发育程度具有显著差异。在抗病玉米材料,病菌初生菌丝、吸器母细胞、次生菌丝的形成时间推迟,吸器少,菌丝分枝少,菌丝生长缓慢。
3抗感自交系经过接种处理后,可溶性蛋白含量都有所下降,与抗病性之间无明显相关性。感病自交系体内可溶性糖含量始终高于抗病自交系。游离脯氨酸含量与苯丙氨酸解氨酶(PAL)酶活在抗病自交系体内增加幅度高于感病自交系。多酚氧化酶(PPO)酶活在抗感自交系体内变化规律与抗病性之间无明显的相关性。抗病自交系体内超氧化物歧化酶(SOD)与过氧化物酶(POD)酶活增幅高于感病自交系,且能长时间维持在较高水平。抗病自交系体内过氧化氢酶(CAT)酶活始终高于对照和感病自交系。因此在玉米苗期,其叶片内可溶性糖和游离脯氨酸含量、PAL、SOD、POD及CAT酶活可作为玉米抗南方锈病的生化指标。
Southern corn rust, caused by Puccinia polysora Underw., is becoming serious in recent years and is a potential threat for maize production in China. With the purpose of studying on the epidemic regularity and resistance mechanism of southern corn rust, specific detection of the pathogen was carried out, and the histopathological characters of P. polysora during infection and colonization in four maize inbred lines were investigated by trypan blue clearing staining technique. Meanwhile, the dynamic change of biochemical indicators was determined. The biochemical indicators associated with southern corn rust resistance were figured out. The main results of this study were as follows:
1. Southern corn rust DNA was amplified by universal primer, and the ITS sequences were obtained after gel extraction, cloning, and sequencing. The length ITS was 692 bp. Primers specific for P. polysora were designed using the primer-design software primer 5. The specific primers were used to amplify some fungal pathogens of maize and healthy tissues. A fragments 365 bp in length was amplified from the pathogen P. polysora only. The detection sensitivity was 10 pg/μL DNA.
2. The histopathological characters of P. polysora during infection and colonization in four maize inbred lines were investigated by trypan blue clearing staining technique. The process of pathogen invasion included five phases: spore germination and germ tube formation, appressorium formation, infection, formation of haustorium, and expansion of mycelium. The differences between spore germination and germ tube information were not very distinct. On the countary, when the pathogen invaded into the host cells of resistant maize lines, the growth and expansion degree of hyphae were distinguished. In the resistant lines, it was later to form primary hyphae, haustoriums and secondery hyphae, and less of haustoria in cells, fewer branches of hyphae and lower growth of hyphae than that in the susceptable lines.
3. After inoculation, the soluble protein contents in resistant and susceptible inbred lines were reduced, which were not associated with disease resisitance. The dissoluble sugar content was higher in the susceptible inbred lines than that in the resistant inbred lines. The resistant inbred lines showed higher increasing rate of the free proline content and the activity of PAL than the susceptible inbred lines. There was no certain correlation on resisitance of the activity of PPO in resistant and susceptible inbred lines. The resistant inbred lines showed higher increasing rate of the activity of SOD and POD. Furthermore, the activity of SOD and POD maintained high level throughout the study. The activity of CAT of the resistant inbred lines was always higher than the susceptible inbred lines and the control. Therefore, at the seedling stage of maize, the dissoluble sugar and the free proline contents and the activity of PAL, SOD, POD, and CAT in leaves can be used as the indictors for screening resistance to P. polysora in maize.
引文
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