甘蔗梢腐病病原分子检测及甘蔗组合、品种的抗病性评价
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摘要
镰刀菌(Gibberella fujikuroi(Saw.) Wholle)无性阶段为Fusarium moniliforme Sheldon,由其引起的甘蔗梢腐病,是甘蔗生长中期最主要的一种真菌性病害。近年来,梢腐病在我国蔗区的发生呈逐渐加重的趋势,对我国甘蔗生产和糖业可持续性发展构成严重威胁。
首先,本研究以50个甘蔗杂交组合的实生苗和13个区试品种为试验材料,在田间自然诱发条件下,分析了甘蔗F1群体和区试品种对梢腐病的抗性。结果显示:13个品种分为三种抗性类型,其中,云蔗99-91属于0级的占了100%,发病率为0。ROC16、粤甘18号、福农02-3924三个品种的调查结果中感病类型相对与其他品种较多,福农02-3924发病率为13.89%,;粤甘18号、ROC16发病率分别为9.44%、8.88%。此外,50个杂交组合分为四种类型,云蔗89-351×CP84-1198组合后代田间自然发病率为0;粤糖00-236×ROC22 ,福农91-4710×ROC10,Hocp93-746×云瑞03-806等4个组合后代实生苗田间自然发病率在0.83%-1.67%之间,占全部组合的8%;福农90-6652×云瑞05-578组合后代实生苗田间自然发病率为15.80%,少数植株出现了梢腐现象;其它45个组合后代的田间自然发病率在2.50%-10.00%之间,占全部组合的90%。
其次,本研究采用ITS-PCR、ATP-PCR和Effd-PCR三种方法获得不同甘蔗品种梢腐病菌相应目的基因片段;而后通过多重序列比对,分析了不同来源菌株的系统进化关系;最后根据ITS序列设计PCR检测特异性引物,构建了甘蔗梢腐病快速检测体系。本研究采集来自我国广东,广西,福建三地感染梢腐病的甘蔗品种上的病斑叶片,接种培养获得病原菌菌落,经光学显微镜和扫描电镜进行形态学观察,分离病原菌单孢,培养后获得相应菌丝体,提取基因组DNA,而后利用ITS-PCR、ATP-PCR和Effd-PCR进行分子鉴定。结果表明,甘蔗梢腐病菌ITS序列与Gibberella moniliformis(甘蔗梢腐病菌的有性态)具有100%同源性,与Gibberella fujikuroi具有99%的同源性,而Effd片段与Fusarium fujikuroi的同源性也高达92%,基于甘蔗梢腐病菌ITS-PCR和Effd-PCR的鉴定结果与电子显微镜和扫描电镜镜的鉴定结果高度符合。相比较而言,ATP较难区分甘蔗梢腐病菌以及其它近缘真菌,ATP片段与F.oxysporum相比仅有79%的同源性。上述研究结果对于甘蔗抗梢腐病育种工作具有一定的指导意义。
Pokkah Boeng disease of sugarcane by Gibberella fujikuroi (Saw.) Wholle causes serious damage to sugarcane industry all over the world. Recently, Pokkah Boeng Disease of sugarcane became a serious threat to the sugarcane production and sugar development in China.
Using the seedings from 50 cross combinations and 13 sugarcane varieties as plant material, the resistance of sugarcane to Pokkah Boeng Disease was evaluated according to rating scales under natural field infection. The results indicated that the infection ratings of the 13 sugarcane varieties were as follows: the infection of YZ99-91 is 0. The corresponding infection rate of FN02-3924, YG18, ROC16 was 13.89%, 9.44% and 8.88% respectively. And the results also showed that the seedings from 50 cross combinations should be classified into four types, The infection rate of the 4 combinationgs (YT00-236×ROC22, Hocp93-746×YR03-806, FN91-4710×ROC10 et al.) was 0.83%-1.67% and this group accounted for 8 percent of all the 50 tested cross combinationgs. The infection rate of the other 45 combinationgs (CP84-1198×GT91-116, CP84-1198×YZ94-375 et al.) was 2.50%-10.00% and this group accounted for 90 percent of all the 50 tested cross combinationgs. Besides, the group of FN90-6652 was 15.80%.
Research on the isolation, culture and identification of the corresponding pathogen should be helpful to the establishment of the fast identification system and aid to sugarcane Pokkah Boeng disease resistance breeding. In this paper, we cut and inoculation-culture the spot leaves from sugarcane cultivar infected by Gibberella fujikuroi(Saw.)Wholle culture which come from the province of Guangdong, Guangxi and Fujian, and the electron microscope and scanning electron microscopy for morphological observation of the corresponding fungi colonies were also conducted, then the separation of single-spore pathogens, the extraction of its DNA and the molecular identification by ITS-PCR, ATP-PCR and Effd-PCR were conducted. We get the targeted gene fragments of different pathogenic fungi from different sugarcane varieties by the methods of ITS-PCR, ATP-PCR and Effd-PCR. The results of bioinformatics analysis showed that the ITS sequence amplified from the above pathogen was 100% homologous with that of Gibberella moniliformis, 99% homologous with that of Gibberella fujikuroi, which demonstrated that the outcome of ITS-PCR and Effd-PCR identification existed a high degree of compliance with the observation through electron microscope and scanning electron microscope. Then, specific PCR primers were designed according to the ITS sequence and a rapid detection system of sugarcane Pokkah Boeng was established. Comparatively speaking, ATP-PCR is relatively more difficult to distinguish sugarcane Pokkah Boeng and its relative fungus. The results above is meaningful to the breeding of surgarcane variety resistant to Pokkah Boeng.
首先,本研究以50个甘蔗杂交组合的实生苗和13个区试品种为试验材料,在田间自然诱发条件下,分析了甘蔗F1群体和区试品种对梢腐病的抗性。结果显示:13个品种分为三种抗性类型,其中,云蔗99-91属于0级的占了100%,发病率为0。ROC16、粤甘18号、福农02-3924三个品种的调查结果中感病类型相对与其他品种较多,福农02-3924发病率为13.89%,;粤甘18号、ROC16发病率分别为9.44%、8.88%。此外,50个杂交组合分为四种类型,云蔗89-351×CP84-1198组合后代田间自然发病率为0;粤糖00-236×ROC22 ,福农91-4710×ROC10,Hocp93-746×云瑞03-806等4个组合后代实生苗田间自然发病率在0.83%-1.67%之间,占全部组合的8%;福农90-6652×云瑞05-578组合后代实生苗田间自然发病率为15.80%,少数植株出现了梢腐现象;其它45个组合后代的田间自然发病率在2.50%-10.00%之间,占全部组合的90%。
其次,本研究采用ITS-PCR、ATP-PCR和Effd-PCR三种方法获得不同甘蔗品种梢腐病菌相应目的基因片段;而后通过多重序列比对,分析了不同来源菌株的系统进化关系;最后根据ITS序列设计PCR检测特异性引物,构建了甘蔗梢腐病快速检测体系。本研究采集来自我国广东,广西,福建三地感染梢腐病的甘蔗品种上的病斑叶片,接种培养获得病原菌菌落,经光学显微镜和扫描电镜进行形态学观察,分离病原菌单孢,培养后获得相应菌丝体,提取基因组DNA,而后利用ITS-PCR、ATP-PCR和Effd-PCR进行分子鉴定。结果表明,甘蔗梢腐病菌ITS序列与Gibberella moniliformis(甘蔗梢腐病菌的有性态)具有100%同源性,与Gibberella fujikuroi具有99%的同源性,而Effd片段与Fusarium fujikuroi的同源性也高达92%,基于甘蔗梢腐病菌ITS-PCR和Effd-PCR的鉴定结果与电子显微镜和扫描电镜镜的鉴定结果高度符合。相比较而言,ATP较难区分甘蔗梢腐病菌以及其它近缘真菌,ATP片段与F.oxysporum相比仅有79%的同源性。上述研究结果对于甘蔗抗梢腐病育种工作具有一定的指导意义。
Pokkah Boeng disease of sugarcane by Gibberella fujikuroi (Saw.) Wholle causes serious damage to sugarcane industry all over the world. Recently, Pokkah Boeng Disease of sugarcane became a serious threat to the sugarcane production and sugar development in China.
Using the seedings from 50 cross combinations and 13 sugarcane varieties as plant material, the resistance of sugarcane to Pokkah Boeng Disease was evaluated according to rating scales under natural field infection. The results indicated that the infection ratings of the 13 sugarcane varieties were as follows: the infection of YZ99-91 is 0. The corresponding infection rate of FN02-3924, YG18, ROC16 was 13.89%, 9.44% and 8.88% respectively. And the results also showed that the seedings from 50 cross combinations should be classified into four types, The infection rate of the 4 combinationgs (YT00-236×ROC22, Hocp93-746×YR03-806, FN91-4710×ROC10 et al.) was 0.83%-1.67% and this group accounted for 8 percent of all the 50 tested cross combinationgs. The infection rate of the other 45 combinationgs (CP84-1198×GT91-116, CP84-1198×YZ94-375 et al.) was 2.50%-10.00% and this group accounted for 90 percent of all the 50 tested cross combinationgs. Besides, the group of FN90-6652 was 15.80%.
Research on the isolation, culture and identification of the corresponding pathogen should be helpful to the establishment of the fast identification system and aid to sugarcane Pokkah Boeng disease resistance breeding. In this paper, we cut and inoculation-culture the spot leaves from sugarcane cultivar infected by Gibberella fujikuroi(Saw.)Wholle culture which come from the province of Guangdong, Guangxi and Fujian, and the electron microscope and scanning electron microscopy for morphological observation of the corresponding fungi colonies were also conducted, then the separation of single-spore pathogens, the extraction of its DNA and the molecular identification by ITS-PCR, ATP-PCR and Effd-PCR were conducted. We get the targeted gene fragments of different pathogenic fungi from different sugarcane varieties by the methods of ITS-PCR, ATP-PCR and Effd-PCR. The results of bioinformatics analysis showed that the ITS sequence amplified from the above pathogen was 100% homologous with that of Gibberella moniliformis, 99% homologous with that of Gibberella fujikuroi, which demonstrated that the outcome of ITS-PCR and Effd-PCR identification existed a high degree of compliance with the observation through electron microscope and scanning electron microscope. Then, specific PCR primers were designed according to the ITS sequence and a rapid detection system of sugarcane Pokkah Boeng was established. Comparatively speaking, ATP-PCR is relatively more difficult to distinguish sugarcane Pokkah Boeng and its relative fungus. The results above is meaningful to the breeding of surgarcane variety resistant to Pokkah Boeng.
引文
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