利用实时荧光定量PCR和数字PCR检测鉴定水稻细菌性条斑病菌  

Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR

作　　者：张健男 王依名 张洁净 陈磊[2] 罗金燕[2] 易建平 李斌[1] 安千里 ZHANG Jiannan;WANG Yiming;ZHANG Jiejing;CHEN Lei;LUO Jinyan;YI Jianping;LI Bin;AN Qianli(State Key Laboratory of Rice Bioogy/Ministry of Agricuture and Rura Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects/Zhejiang Provincial Key Laboratory of Biology of Crop Pathogens and Insects/College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,Zhejiang,China;Shanghai Agriculture Technology Extension and Service Center,Shanghai 201103,China;Technical Center for Animal,Plant and Food Inspection and Quarantine of Shanghai Customs District,Shanghai 200135,China)

机构地区：[1]水稻生物学国家重点实验室/农业农村部作物病虫分子生物学重点实验室/浙江省作物病虫生物学重点实验室/浙江大学农业与生物技术学院,浙江杭州310058 [2]上海市农业技术推广服务中心,上海201103 [3]上海海关动植物与食品检验检疫技术中心,上海200135

出　　处：《浙江大学学报（农业与生命科学版）》2023年第1期55-64,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：上海市科技兴农项目(2019-02-08-00-08-F01150);浙江省重点研发计划项目(2019C02006)。

摘　　要：白叶枯病和条斑病是水稻重要的细菌性病害。水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)和水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)属于同种下的2个致病变种。鉴别Xoo和Xoc对检疫和防控这2种病害至关重要。Xoo和Xoc中的铁-红酵母酸/铁-粪生素受体基因fhuE在进化过程中因不同程度地缺失而成为假基因。针对Xoc中有而Xoo中缺失的fhuE部分序列设计引物,筛选出Xoc特异引物XocFhuE-F(5’-ATCGAACGATGTCACCAGGG-3’)和XocFhuE-R(5’-AGAAACGTGCGGCCAGATAA-3’)。用XocFhuE-F/XocFhuE-R能只从Xoc菌株中仅扩增出159 bp片段,并结合荧光染料建立了SYBR Green实时荧光定量PCR(quantitative real-time PCR,qPCR)和EvaGreen微滴数字PCR(digital PCR,dPCR)方法来检测鉴定Xoc。在20μL反应体系中加1μL模板,qPCR检测菌悬液中Xoc的下限是1.6×10^(4)CFU/mL,检测带菌种子中Xoc的下限是1.2×10^(3)CFU/粒;d PCR检测菌悬液中Xoc的下限是1.6×10^(3)CFU/mL,检测带菌种子中Xoc的下限是1.2×10^(2)CFU/粒。综上所述,基于Xoc特异引物XocFhuE-F/XocFhuE-R建立的SYBR Green qPCR和EvaGreen dPCR为检疫Xoc和监测预警水稻条斑病提供了高效的检测方法。Bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae(Xoo) and bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola(Xoc) are two important bacterial diseases of rice.Identification of Xoo and Xoc belonging to the same species is critical for quarantine and control the diseases.Here, we found that the gene fhuE encoding ferric-rhodotorulic acid/ferric-coprogen receptor in Xoo and Xoc became pseudogenes after partial deletion during evolution. We designed Xoc-specific primers XocFhuE-F(5’-ATCGAACGATGTCACCAGGG-3’) and XocFhuE-R(5’-AGAAACGTGCGGCCAGATAA-3’) targeting the sequences present in the Xoc fhuE pseudogene but absent in the Xoo fhuE pseudogene. Polymerase chain reaction(PCR) amplification produced only a 159 bp DNA fragment from only Xoc strains using the primers XocFhuE-F/XocFhuE-R. Based on the Xoc-specific primers, we developed SYBR Green quantitative real-time PCR(qPCR) and EvaGreen droplet digital PCR(dPCR) methods to detect and identify Xoc. In a 20 μL reaction system with 1 μL of template, the detection limit of qPCR on Xoc was 1.6×10^(4) CFU/mL in bacterial suspension and 1.2×10^(3) CFU/seed in rice seeds;the detection limit of dPCR on Xoc was 1.6×10^(3) CFU/mL in bacterial suspension and 1.2×10^(2) CFU/seed in rice seeds. In conclusion, SYBR Green qPCR and EvaGreen dPCR based on the Xoc-specific primers XocFhuE-F/XocFhuE-R provide effective methods for quarantine of Xoc and monitoring of the bacterial leaf streak disease of rice.

关 键 词：水稻条斑病 水稻白叶枯病 水稻黄单胞菌 保守标志蛋白 铁-粪生素受体 

分 类 号：S41-30[农业科学—植物保护]