一种lamp技术快速检测抗甲基硫菌灵的草坪币斑病菌突变位点方法的建立与应用
币斑病菌（Sclerotinia homoeocarpa F.T. Bennett）可以广泛地侵染冷、暖季型草坪草，引起草坪的币斑病病害，对草坪景观及其生态、经济效益造成极大破坏。目前，化学防治是被草坪管理业广泛采用的防治方法，但长期使用较为固定的杀菌剂已使多种病原真菌产生了不同程度抗药性，尤其是币斑病菌，其对主流杀菌剂，如甲基硫菌灵的抗药性突变，大幅降低了其对币斑病防治的效果。环介导等温扩增（LAMP）技术尚未应用于抗甲基硫菌灵的草坪币斑病菌检测，这一灵敏度高、特异性强、操作简便的新方法，在该领域有极大应用前景。
目录
摘要1
关键词
1
Abstract
1
引言
1
引言2
1材料与方法5
1.1供试币斑病菌菌株
5
1.2实验所需试剂与仪器
6
1.3抗甲基硫菌灵的草坪币斑病菌突变位点LAMP引物的设计与合成
6
1.4币斑病菌DNA的提取
6
1.5 LAMP反应体系的优化
6
2结果与分析
7
2.1特异性LAMP体系的扩增
7
2.2 LAMP体系中dNTPs对反应的影响
7
2.3 LAMP体系中Mg2+对反应的影响
8
2.4 LAMP体系中甜菜碱（betaine）对反应的影响
8
2.5检测币斑病菌的LAMP预设体系的建立
9
2.6检测抗甲基硫菌灵的草坪币斑病菌突变位点的LAMP体系建立
9
3讨论10
致谢
11
参考文献
12
图1 dNTPs的浓度优化
7
图2 Mg2+的浓度优化
8
图3 甜菜碱的浓度优化
8
图4 设计的3套引物筛选结果
9
表1本文所用菌株信息
*好棒文|www.hbsrm.com +Q: ^351916072*
6
表2本文所用LAMP引物
9
一种基于LAMP技术快速检测抗甲基硫菌灵的草坪币斑病菌突变位点方法的建立与应用
Establishment and application of a loopmediated isothermal amplification method for detection of Thiophanatemethyl resistant mutant in Sclerotinia homoeocarpa F.T. Bennett
Student majoring in Grassland Science Jingya Yang
Tutor Jian Hu
Abstract：Sclerotinia homoeocarpa F.T. Bennett is a fungal pathogen which causes Dollar Spot, a kind of plant disease of turfgrass, on lawn universally in the world. The fungus infects grass comprehensively, for example, a large scale of varieties of coolseason grass and warmseason grass can be infected. Dollar Spot leads to a terrible expense in the landscape, together with the ecologic and economic value of turfgrass, because the disease damages the scene and balance in the environment surrounding the grass. In order to control the effects of the disease, chemical fungicide is the most popular method in management nowadays. However, isolates with resistance to popular fungicide are discovered currently as the consequence of these chemicals’ frequent application. S. homoeocarpa is a typical instance of Thiophanatemethyl resistant pathogenic fungi on turfgrass, which terribly reduce the efficiency of thiophanatemethyl and, because of the potential multiple resistance existed in isolates, other benzimidazole chemicals are possible to have difficulties in managing dollar spot as well. Loopmediated isothermal amplification (LAMP) is a novel assay for detection of fungicideresistant isolates of fungi, since its sensitivity, specificity, simplification and efficiency are superior than the majority of detection approaches reported. With the application of LAMP, the efficiency of turfgrass management will possibly be promoted.
目录
摘要1
关键词
1
Abstract
1
引言
1
引言2
1材料与方法5
1.1供试币斑病菌菌株
5
1.2实验所需试剂与仪器
6
1.3抗甲基硫菌灵的草坪币斑病菌突变位点LAMP引物的设计与合成
6
1.4币斑病菌DNA的提取
6
1.5 LAMP反应体系的优化
6
2结果与分析
7
2.1特异性LAMP体系的扩增
7
2.2 LAMP体系中dNTPs对反应的影响
7
2.3 LAMP体系中Mg2+对反应的影响
8
2.4 LAMP体系中甜菜碱（betaine）对反应的影响
8
2.5检测币斑病菌的LAMP预设体系的建立
9
2.6检测抗甲基硫菌灵的草坪币斑病菌突变位点的LAMP体系建立
9
3讨论10
致谢
11
参考文献
12
图1 dNTPs的浓度优化
7
图2 Mg2+的浓度优化
8
图3 甜菜碱的浓度优化
8
图4 设计的3套引物筛选结果
9
表1本文所用菌株信息
*好棒文|www.hbsrm.com +Q: ^351916072*
6
表2本文所用LAMP引物
9
一种基于LAMP技术快速检测抗甲基硫菌灵的草坪币斑病菌突变位点方法的建立与应用
Establishment and application of a loopmediated isothermal amplification method for detection of Thiophanatemethyl resistant mutant in Sclerotinia homoeocarpa F.T. Bennett
Student majoring in Grassland Science Jingya Yang
Tutor Jian Hu
Abstract：Sclerotinia homoeocarpa F.T. Bennett is a fungal pathogen which causes Dollar Spot, a kind of plant disease of turfgrass, on lawn universally in the world. The fungus infects grass comprehensively, for example, a large scale of varieties of coolseason grass and warmseason grass can be infected. Dollar Spot leads to a terrible expense in the landscape, together with the ecologic and economic value of turfgrass, because the disease damages the scene and balance in the environment surrounding the grass. In order to control the effects of the disease, chemical fungicide is the most popular method in management nowadays. However, isolates with resistance to popular fungicide are discovered currently as the consequence of these chemicals’ frequent application. S. homoeocarpa is a typical instance of Thiophanatemethyl resistant pathogenic fungi on turfgrass, which terribly reduce the efficiency of thiophanatemethyl and, because of the potential multiple resistance existed in isolates, other benzimidazole chemicals are possible to have difficulties in managing dollar spot as well. Loopmediated isothermal amplification (LAMP) is a novel assay for detection of fungicideresistant isolates of fungi, since its sensitivity, specificity, simplification and efficiency are superior than the majority of detection approaches reported. With the application of LAMP, the efficiency of turfgrass management will possibly be promoted.
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