山东省十字花科蔬菜黑腐病菌的鉴定及大白菜品种抗病性研究
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摘要
白菜黑腐病由野油菜黄单胞杆菌Xanthomonas campestris引起,是一种世界性十字花科蔬菜重要病害。本研究论文从白菜黑腐病的采集分离研究开始,分别进行了白菜黑腐病的鉴定、白菜对黑腐病抗性鉴定方法的探索、品种抗病性的鉴定以及白菜接种黑腐病菌后防御酶系的变化分析。所得结果概括如下:
1. 2005-2006年,从山东泰安、济南、烟台、威海等地采集白菜黑腐病病害标样25份,用平板划线法室内分离纯化获得黑腐病菌65株。经致病性测定,选出四个强致病力菌株H9、C2、E3、G2,分别对其进行了形态学观察、生物学特性测定和分子检测。四个菌株其生物学特性反应表现基本一致,16S-ITSrDNA序列分析表明供试病原菌与Genbank中的AE12540.1 (Xanthomonas campestris pv. campestris)的同源性是99.63%。结合形态学特征和致病性反应,确认引起山东省白菜黑腐病的病原菌是野油菜黄单胞杆菌野油菜黑腐病致病变种(Xanthomonas campestris pv. campestris)。
2.通过采用不同的接种方法、接种浓度和接种苗龄等对白菜黑腐病苗期抗病性进行了测定,对其表现进行比较,筛选出一套适用于白菜苗期的快速准确的抗病性鉴定方法。白菜黑腐病苗期抗病性鉴定,采用3×108cfu/mL的病原菌悬浮液,在幼苗3～4片真叶期进行“喷雾法”接种,能较好地表现发病情况,接种后第七天表现典型症状,反映不同材料对黑腐病的抗病性差异。
3.通过37个大白菜品种对黑腐病的抗病性鉴定结果表明,供试品种均未表现免疫反应,不同大白菜品种的抗病性有一定的差异。高抗品种有改良83-24、德高1号和正旺达12,病情指数在30以下,占供试总品种的8.1%;中抗品种有夏阳、天正品优等,占总品种的56.8%;强春、四季王、胶白8号等表现感病,中感占27.0%;高感品种有橘红60、金秀、北京小杂56,病情指数都在70以上,占供试品种的8.1%。
4.在37份白菜材料中选取“抗病”、“感病”不同抗病性级别的材料6份,分别进行黑腐病病原菌的接种处理和未接种(喷蒸馏水)处理,并测定接种前后幼苗叶片内的SOD酶、POD酶、CAT酶、PPO酶和PAL酶等5种酶活性的变化,分析这些变化与其抗病性的关系。
Cabbage Black Rot disease is caused by Xanthomonas campestris , that is a major disease in cruciferous vegetables worldwide. This research paper study on the Separation of disease leaves about the cabbage Black Rot, then we conducted identification of cabbage Black Rot disease , exploration of methods of identification of resistance on Chinese cabbage to Black Rot disease, the identification of resistance on varieties of cabbage and the analysis of changes on defence Enzymes after inoculate Black Rot on Cabbages. The results can be summarized as follows:
1. We collected 25 samples of Chinese cabbage Black Rot from different fields of Shandong Province during 2005 ~ 2006 years , such as Taian, Jinan, Yantai,Weihai and so on . With that we gained 65 bacterial strains after separation and purity in the lab. Four bacterial strains were voted in pathogenicity mensurate. morphology observation, Biology characteristic and molecules measure of the four strains (named H9、C2、E3、G2) revealed that Biology characteristic reaction shows four examinated bacterial strain coinciding basically , 16S-ITSrDNA sequence analysis indicates the homologous identity between Xanthomonas campestris pv. campestris in Genbank and pathogenic bacteria is 99.63%. The pathogenic bacteria which arouse Chinese cabbage Black Rot in Shandong Province belong to Xanthomonas campestris pv. campestris.
2. Through the use of testing different inoculating methods,inoculated concentrations and Age of Chinese cabbage of vaccination, and other black rot disease resistance seedlings were determined to compare their performance,after that screening a set of rapid and accurate methods of identification of resistance to cabbage seedlings. Identification of resistance of black rot on Cabbage seedlings , That is while in the temperatures ranged from 25℃to 30℃, the suspensions of 3×108 colony forming units/mL were sprayed to the leafs seedlings with four to five, the moisture was kept for 24 to 48 hours.That can be better performance of the situation , In seven days after treatmented , leaves performance typical symptoms,reflect the different resistance to black rot on different materials. It was showed that the technique could be easy to distinguish the resistant differences in different varieties of xanthomonas campestris.
3. Through identification of resistance on the 37 varieties of Chinese cabbage in Shandong Province to black rot with this method indicated, results showed that all the performance of the tested varieties were not immune response, the resistance of different varieties of Chinese cabbage represent a certain difference. The percentage of high resistant varieties such as Improved varieties 83-24 , Degao.1 and Zhengwd.12 have high resistance , the following condition index of 30, accounting for the trial of species of 8.1%;The moderate resistant varieties w Xia Yang, Tianzheng Excellence and so on, accounting for the total species of 56.8% ; The disease susceptible varieties Qiangchun, Sijiwang, Jiaobai.8 and other performance flu disease was 27.0% ; highly susceptible varieties Juhong60, Jinxiu, Beijingxiaza56 in the condition index above 70, accounting for the total species of 8.1%.
4. We selected resistance and flu disease resistance levels of different materials 6 shares of 37 Cabbage material, after dealed with Black Rot disease pathogens vaccination and not vaccinated (spray distilled water) ,and determine the activity changes of the five kinds of Enzyme before and after vaccination Leaves,such as superoxide dismutase,peroxidase,catalase,polyphenol oxidase,phenylalanine ammonia lyase,then analysis the relationship between the changes and the resistance.
1. 2005-2006年,从山东泰安、济南、烟台、威海等地采集白菜黑腐病病害标样25份,用平板划线法室内分离纯化获得黑腐病菌65株。经致病性测定,选出四个强致病力菌株H9、C2、E3、G2,分别对其进行了形态学观察、生物学特性测定和分子检测。四个菌株其生物学特性反应表现基本一致,16S-ITSrDNA序列分析表明供试病原菌与Genbank中的AE12540.1 (Xanthomonas campestris pv. campestris)的同源性是99.63%。结合形态学特征和致病性反应,确认引起山东省白菜黑腐病的病原菌是野油菜黄单胞杆菌野油菜黑腐病致病变种(Xanthomonas campestris pv. campestris)。
2.通过采用不同的接种方法、接种浓度和接种苗龄等对白菜黑腐病苗期抗病性进行了测定,对其表现进行比较,筛选出一套适用于白菜苗期的快速准确的抗病性鉴定方法。白菜黑腐病苗期抗病性鉴定,采用3×108cfu/mL的病原菌悬浮液,在幼苗3～4片真叶期进行“喷雾法”接种,能较好地表现发病情况,接种后第七天表现典型症状,反映不同材料对黑腐病的抗病性差异。
3.通过37个大白菜品种对黑腐病的抗病性鉴定结果表明,供试品种均未表现免疫反应,不同大白菜品种的抗病性有一定的差异。高抗品种有改良83-24、德高1号和正旺达12,病情指数在30以下,占供试总品种的8.1%;中抗品种有夏阳、天正品优等,占总品种的56.8%;强春、四季王、胶白8号等表现感病,中感占27.0%;高感品种有橘红60、金秀、北京小杂56,病情指数都在70以上,占供试品种的8.1%。
4.在37份白菜材料中选取“抗病”、“感病”不同抗病性级别的材料6份,分别进行黑腐病病原菌的接种处理和未接种(喷蒸馏水)处理,并测定接种前后幼苗叶片内的SOD酶、POD酶、CAT酶、PPO酶和PAL酶等5种酶活性的变化,分析这些变化与其抗病性的关系。
Cabbage Black Rot disease is caused by Xanthomonas campestris , that is a major disease in cruciferous vegetables worldwide. This research paper study on the Separation of disease leaves about the cabbage Black Rot, then we conducted identification of cabbage Black Rot disease , exploration of methods of identification of resistance on Chinese cabbage to Black Rot disease, the identification of resistance on varieties of cabbage and the analysis of changes on defence Enzymes after inoculate Black Rot on Cabbages. The results can be summarized as follows:
1. We collected 25 samples of Chinese cabbage Black Rot from different fields of Shandong Province during 2005 ~ 2006 years , such as Taian, Jinan, Yantai,Weihai and so on . With that we gained 65 bacterial strains after separation and purity in the lab. Four bacterial strains were voted in pathogenicity mensurate. morphology observation, Biology characteristic and molecules measure of the four strains (named H9、C2、E3、G2) revealed that Biology characteristic reaction shows four examinated bacterial strain coinciding basically , 16S-ITSrDNA sequence analysis indicates the homologous identity between Xanthomonas campestris pv. campestris in Genbank and pathogenic bacteria is 99.63%. The pathogenic bacteria which arouse Chinese cabbage Black Rot in Shandong Province belong to Xanthomonas campestris pv. campestris.
2. Through the use of testing different inoculating methods,inoculated concentrations and Age of Chinese cabbage of vaccination, and other black rot disease resistance seedlings were determined to compare their performance,after that screening a set of rapid and accurate methods of identification of resistance to cabbage seedlings. Identification of resistance of black rot on Cabbage seedlings , That is while in the temperatures ranged from 25℃to 30℃, the suspensions of 3×108 colony forming units/mL were sprayed to the leafs seedlings with four to five, the moisture was kept for 24 to 48 hours.That can be better performance of the situation , In seven days after treatmented , leaves performance typical symptoms,reflect the different resistance to black rot on different materials. It was showed that the technique could be easy to distinguish the resistant differences in different varieties of xanthomonas campestris.
3. Through identification of resistance on the 37 varieties of Chinese cabbage in Shandong Province to black rot with this method indicated, results showed that all the performance of the tested varieties were not immune response, the resistance of different varieties of Chinese cabbage represent a certain difference. The percentage of high resistant varieties such as Improved varieties 83-24 , Degao.1 and Zhengwd.12 have high resistance , the following condition index of 30, accounting for the trial of species of 8.1%;The moderate resistant varieties w Xia Yang, Tianzheng Excellence and so on, accounting for the total species of 56.8% ; The disease susceptible varieties Qiangchun, Sijiwang, Jiaobai.8 and other performance flu disease was 27.0% ; highly susceptible varieties Juhong60, Jinxiu, Beijingxiaza56 in the condition index above 70, accounting for the total species of 8.1%.
4. We selected resistance and flu disease resistance levels of different materials 6 shares of 37 Cabbage material, after dealed with Black Rot disease pathogens vaccination and not vaccinated (spray distilled water) ,and determine the activity changes of the five kinds of Enzyme before and after vaccination Leaves,such as superoxide dismutase,peroxidase,catalase,polyphenol oxidase,phenylalanine ammonia lyase,then analysis the relationship between the changes and the resistance.
引文
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