PDF《番茄细菌性斑点病菌、 溃疡病菌、青枯病菌和疮》

园艺学报,2018,45 (11):2254C2264.

Acta Horticulturae Sinica

2254 doi:10.16420/j.issn.0513-353x.2018-0153;

http://www. ahs. ac. cn 收稿日期:2018C04C25;

修回日期:2018C11C20 基金项目:国家重点研发计划项目(2017YFD0200603) ;

中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS) ;

农业部园艺作 物生物学与种质创制重点实验室 * 通信作者 Author for correspondence(E-mail:[email protected];

[email protected]) 番茄细菌性斑点病菌、 溃疡病菌、 青枯病菌和疮 痂病菌的四重 PCR 检测方法 康华军 1,2 ,柴阿丽 1,* ,石延霞

1 ,谢学文

1 ,袁军海

2 ,李宝聚 1,* (1 中国农业科学院蔬菜花卉研究所,北京 100081;

2 河北北方学院,河北张家口 075061) 摘要:针对引起番茄细菌性病害的细菌性斑点病菌(Pseudomonas syringae pv. tomato,Pst)、溃 疡病菌(Clavibacter michiganensis subsp. michiganensis,Cmm)、青枯病菌(Ralstonia solanacearum,Rs) 以及疮痂病菌(Xanthomonas campestris pv. vesicatoria,Xcv),建立了四重 PCR 检测技术,为病害的快 速、准确诊断提供技术支持.根据 gap1 基因设计 Pst 特异性引物 BW-F/BW-R,经PCR 条件优化,扩增 出了

375 bp 的特异性片段.将设计的引物与已报道的

3 种细菌特异性引物组合,通过设定不同的退火温 度、引物浓度、循环次数以及延伸时间,探索影响四重 PCR 扩增的因素,优化了其反应体系.四重 PCR 反应体系中的引物对 BW-F/BW-R、Fan1/Fan

2、RS-1-F/RS-3-R 和XCVF/XCVR 可分别扩增出细菌性斑点 病菌、溃疡病菌、青枯病菌和疮痂病菌长度为

375、

146、716 和517 bp 的特异性目的片段,反应体系退 火温度为 57.1 ℃,4 对引物的终浓度分别为 0.

24、0.

16、0.16 和0.08 μmol ・ L-1 ,延伸时间

45 s,35 个循 环.该四重 PCR 反应体系可快速检测田间番茄发病植株中的细菌性斑点病菌、溃疡病菌、青枯病菌和疮 痂病菌,灵敏度达到 10-1 ng ・ μL-1 . 关键词:番茄;

细菌性斑点病菌;

溃疡病菌;

青枯病菌;

疮痂病菌;

四重 PCR 中图分类号:S 641.2 文献标志码:A 文章编号:0513-353X(2018)11-2254-11 Quadruple PCR Detection of Pseudomonas syringae pv. tomato,Clavibacter michiganensis subsp. michiganensis,Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria in Infected Tomato Tissues KANG Huajun1,2 ,CHAI Ali1,* ,SHI Yanxia1 ,XIE Xuewen1 ,YUAN Junhai2 ,and LI Baoju1,* (1 Institute of Vegetables and Flowers,Chinese Academy of Agricultural Science,Beijing 100081,China;

2 Hebei North University,Zhangjiakou,Hebei 075061,China) Abstract: In order to establish a rapid and specific detection method for tomato diseases, a quadruple PCR assay was designed for identifing the pathogens including Pseudomonas syringae pv. tomato(Pst), Clavibacter michiganensis subsp. michiganensis (Cmm) , Ralstonia solanacearum (Rs) and Xanthomonas campestris pv. vesicatoria(Xcv). The specific primers(BW-F/BW-R)of Pst were designed based on the gap1 gene sequence,which can amplify

375 bp specific fragment by optimizing the PCR conditions. Besides,three pairs of specific primers of Cmm(Fan1/Fan2),Rs(RS-1-F/RS-3-R),and Xcv(XCVF/ 康华军,柴阿丽,石延霞,谢学文,袁军海,李宝聚. 番茄细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌的四重 PCR 检测方法. 园艺学报,2018,45 (11):2254C2264.

2255 XCVR)used in this study were referenced previous studies. The primer concentration,the annealing temperature, amplification cycles, and the extension time were optimized to obtain the best ratio of primers and amplification conditions. Then,the rapid quadruple PCR detection of pathogens in tomato was established. The annealing temperature was 57.1 ℃,the extension time was