春夏大白菜黑斑病抗性鉴定和抗病机理研究
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摘要
大白菜黑斑病是由链格孢属真菌所致的一种常发病害,也是一种世界性病害。近年来,随着春大白菜、早秋大白菜的发展,黑斑病病害问题日益突出,抗病育种是解决大白菜黑斑病经济而又有效的途径,而抗性鉴定和抗病机理研究则是抗病育种的基础。为此,本研究从春夏大白菜黑斑病病原菌分离、鉴定入手,在研究了大白菜黑斑病苗期抗性鉴定方法的基础上,对168份大白菜材料进行苗期大白菜黑斑病抗性鉴定,同时分析了不同抗性的大白菜品种接种病原菌后POD、CAT、PAL活性的变化。取得的研究结果如下:
1在陕西省大白菜主产区分离得到32个黑斑病病菌单孢系,根据形态和特异引物PCR扩增结果,确定是甘蓝链格孢Alternaria brassicicola。其在PDA人工培养基上适宜生长温度为25～30℃。寄主范围试验表明,甘蓝链格孢可侵染小白菜、萝卜、芜菁、甘蓝、芥蓝、菜心、芥菜、油菜。
2建立了大白菜抗甘蓝链格孢黑斑病的人工接种鉴定方法,其程序为:采用二叶期幼苗,以1×104pfu/mL孢子浓度,喷雾接种,随后置25℃条件下,先黑暗下保湿24h,接着正常管理3天,从第4天开始夜间保湿,白天揭开,第8天保湿24h后,调查发病情况,其结果客观反映大白菜品种的抗病性。
3对46份大白菜杂交组合和122份自交系进行了苗期抗病性鉴定,筛选出30份抗病材料、109份中抗材料、24份感病材料和5份高感材料,未见高抗与免疫品种,为抗病育种和分子标记研究奠定了基础。抗病材料可作为抗病的亲本,其中自交系材料06S157的病情指数仅14.6,说明其对甘蓝链格孢的抗性好,可在以后的育种过程中加以利用。
4抗病品种在受到甘蓝链格孢侵染时,叶片内POD活性反应较感病品种快,但POD活性值上升比感病品种小;抗病品种在受到病原菌侵染时,叶片内CAT活性反应快,且一直维持在一个较高的水平,而感病品种在受病菌侵染后叶片内的CAT活性变化缓慢,变化程度低;另外无论是抗病品种还是感病品种的PAL酶活性在接种后的动态变化有相似规律,接种后所有品种的PAL酶活性均比未接种前增加,但品种之间PAL酶活性没有显著差异。
Black spot disease of Chinese cabbage is caused by Alternaria genus not only frequently occurs in Chinese cabbage, but also widely occurs in the world. As the rapid developing of Chinese cabbage cultivated in spring and early autumn in recent years, the problem of black spot disease was increasing seriously. It is well known that disease-resistant breeding is an economical and effective way to prevent the disease popularity in Chinese cabbage, the resistance identification and disease-resistant mechanism are the foundation of disease-resistant breeding. In this research program, the disease pathogen of black spot disease in spring-summer Chinese cabbage was isolated and identified firstly, then a convenient and feasible resistant-identification method to black spot disease of Chinese cabbage was estabolished at seedling stage, the resistance of 168 Chinese cabbage lines and hybrids to black spot disease were identified at seedling stage, and the change of POD, CAT, PAL enzyme activities in leaves after inoculation were also analyzed. The main results were as follows:
1. 32 single-spore-lines were isolated from the disease leaves in the main Chinese cabbage production areas of Shaanxi Province. It was determined that the A.brassicicola was the only pathogen of it according to the spore’s shape and the profile of PCR amplification with special primers. The suitable temperature for the growth of A.brassicicola on PDA medium was 25～30℃. The host experiment indicated that it can infect Chinese cabbage, radish, turnip, cabbage, Chinese kale, flowering Chinese cabbage, mustard and rapeseed.
2. An artificial inoculation method for black spot disease identification was estabolished, which key procedure is praying 1×104 pfu/mL spores solution on seedling with two true leaves, and placing at 25℃condition, then keeping saturation moisture and dark for 24h, and conventional condition from 2th-4th day, keeping moisture at night and opening at day from 4th-7th day, keeping moisture 24h in 8th day, then investigating disease grade. The result from the method was exact in accordance with the true resistance type of Chinese cabbage.
3. 46 hybrids and 122 inbred lines of Chinese cabbage were appraised for the resistance to black spot disease at seedling stage; the result was that there were 30 resistant materials, 109 medial-resistant materials, 24 susceptible materials and 5 high-susceptible materials. the resistant materials will be used as parent in disease-resistant breeding in future, especially the line of 06S157 which disease index is only 14.6.There was not found any high-resistant and immunity materials in it, which means the road of disease-resistance breeding is difficult.
4. The measurement of antioxidant activities showed that POD activity in the resistant cultivars rised earlier than in the susceptible ones after inoculation with A.brassicicala, but the highest point of POD activity in the resistant cultivars was lower than in the susceptible cultivars; The activities of CAT in the resistant cultivars raised earlier and kept a high level after inoculation with A. brassicicola, but slow and small change in the susceptible cultivars; The dynamic change of PAL activity were similar in susceptible and resistant varieties after inoculation with A.brassicicola, which was that the activities of PAL were increased after inoculation with A.brassicicola, but the activity of PAL had no significant difference between susceptible and resistant cultivars.
1在陕西省大白菜主产区分离得到32个黑斑病病菌单孢系,根据形态和特异引物PCR扩增结果,确定是甘蓝链格孢Alternaria brassicicola。其在PDA人工培养基上适宜生长温度为25～30℃。寄主范围试验表明,甘蓝链格孢可侵染小白菜、萝卜、芜菁、甘蓝、芥蓝、菜心、芥菜、油菜。
2建立了大白菜抗甘蓝链格孢黑斑病的人工接种鉴定方法,其程序为:采用二叶期幼苗,以1×104pfu/mL孢子浓度,喷雾接种,随后置25℃条件下,先黑暗下保湿24h,接着正常管理3天,从第4天开始夜间保湿,白天揭开,第8天保湿24h后,调查发病情况,其结果客观反映大白菜品种的抗病性。
3对46份大白菜杂交组合和122份自交系进行了苗期抗病性鉴定,筛选出30份抗病材料、109份中抗材料、24份感病材料和5份高感材料,未见高抗与免疫品种,为抗病育种和分子标记研究奠定了基础。抗病材料可作为抗病的亲本,其中自交系材料06S157的病情指数仅14.6,说明其对甘蓝链格孢的抗性好,可在以后的育种过程中加以利用。
4抗病品种在受到甘蓝链格孢侵染时,叶片内POD活性反应较感病品种快,但POD活性值上升比感病品种小;抗病品种在受到病原菌侵染时,叶片内CAT活性反应快,且一直维持在一个较高的水平,而感病品种在受病菌侵染后叶片内的CAT活性变化缓慢,变化程度低;另外无论是抗病品种还是感病品种的PAL酶活性在接种后的动态变化有相似规律,接种后所有品种的PAL酶活性均比未接种前增加,但品种之间PAL酶活性没有显著差异。
Black spot disease of Chinese cabbage is caused by Alternaria genus not only frequently occurs in Chinese cabbage, but also widely occurs in the world. As the rapid developing of Chinese cabbage cultivated in spring and early autumn in recent years, the problem of black spot disease was increasing seriously. It is well known that disease-resistant breeding is an economical and effective way to prevent the disease popularity in Chinese cabbage, the resistance identification and disease-resistant mechanism are the foundation of disease-resistant breeding. In this research program, the disease pathogen of black spot disease in spring-summer Chinese cabbage was isolated and identified firstly, then a convenient and feasible resistant-identification method to black spot disease of Chinese cabbage was estabolished at seedling stage, the resistance of 168 Chinese cabbage lines and hybrids to black spot disease were identified at seedling stage, and the change of POD, CAT, PAL enzyme activities in leaves after inoculation were also analyzed. The main results were as follows:
1. 32 single-spore-lines were isolated from the disease leaves in the main Chinese cabbage production areas of Shaanxi Province. It was determined that the A.brassicicola was the only pathogen of it according to the spore’s shape and the profile of PCR amplification with special primers. The suitable temperature for the growth of A.brassicicola on PDA medium was 25～30℃. The host experiment indicated that it can infect Chinese cabbage, radish, turnip, cabbage, Chinese kale, flowering Chinese cabbage, mustard and rapeseed.
2. An artificial inoculation method for black spot disease identification was estabolished, which key procedure is praying 1×104 pfu/mL spores solution on seedling with two true leaves, and placing at 25℃condition, then keeping saturation moisture and dark for 24h, and conventional condition from 2th-4th day, keeping moisture at night and opening at day from 4th-7th day, keeping moisture 24h in 8th day, then investigating disease grade. The result from the method was exact in accordance with the true resistance type of Chinese cabbage.
3. 46 hybrids and 122 inbred lines of Chinese cabbage were appraised for the resistance to black spot disease at seedling stage; the result was that there were 30 resistant materials, 109 medial-resistant materials, 24 susceptible materials and 5 high-susceptible materials. the resistant materials will be used as parent in disease-resistant breeding in future, especially the line of 06S157 which disease index is only 14.6.There was not found any high-resistant and immunity materials in it, which means the road of disease-resistance breeding is difficult.
4. The measurement of antioxidant activities showed that POD activity in the resistant cultivars rised earlier than in the susceptible ones after inoculation with A.brassicicala, but the highest point of POD activity in the resistant cultivars was lower than in the susceptible cultivars; The activities of CAT in the resistant cultivars raised earlier and kept a high level after inoculation with A. brassicicola, but slow and small change in the susceptible cultivars; The dynamic change of PAL activity were similar in susceptible and resistant varieties after inoculation with A.brassicicola, which was that the activities of PAL were increased after inoculation with A.brassicicola, but the activity of PAL had no significant difference between susceptible and resistant cultivars.
引文
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