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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2023, Vol. 49 Issue (1): 55-64    DOI: 10.3785/j.issn.1008-9209.2021.12.271
植物保护     
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
张健男1(),王依名1,张洁净1,陈磊2,罗金燕2,易建平3,李斌1,安千里1()
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
Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR
Jiannan ZHANG1(),Yiming WANG1,Jiejing ZHANG1,Lei CHEN2,Jinyan LUO2,Jianping YI3,Bin LI1,Qianli AN1()
1.State Key Laboratory of Rice Biology/Ministry of Agriculture and Rural 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
2.Shanghai Agriculture Technology Extension and Service Center, Shanghai 201103, China
3.Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs District, Shanghai 200135, China
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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20 μL反应体系中加1 μL模板，qPCR检测菌悬液中Xoc的下限是1.6×104 CFU/mL，检测带菌种子中Xoc的下限是1.2×103 CFU/粒；dPCR检测菌悬液中Xoc的下限是1.6×103 CFU/mL，检测带菌种子中Xoc的下限是1.2×102 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。
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    
Abstract:

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 1.6×104 CFU/mL in bacterial suspension and 1.2×103 CFU/seed in rice seeds; the detection limit of dPCR on Xoc was 1.6×103 CFU/mL in bacterial suspension and 1.2×102 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.
Key words: bacterial leaf streak of rice    bacterial leaf blight of rice    Xanthomonas oryzae    conserved signature protein    ferric-coprogen receptor
收稿日期: 2021-12-27 出版日期: 2023-03-07
CLC:  S41-30  
基金资助: 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)
通讯作者: 安千里     E-mail: 1844978177@qq.com;an@zju.edu.cn
作者简介: 张健男（https://orcid.org/0000-0002-4571-0079），E-mail：1844978177@qq.com
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引用本文:

张健男,王依名,张洁净,陈磊,罗金燕,易建平,李斌,安千里. 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[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(1): 55-64.

Jiannan ZHANG,Yiming WANG,Jiejing ZHANG,Lei CHEN,Jinyan LUO,Jianping YI,Bin LI,Qianli AN. Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(1): 55-64.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.12.271    或    https://www.zjujournals.com/agr/CN/Y2023/V49/I1/55

细菌菌株

Bacterial strain

分离地

Isolation location

菌株保藏来源

Culture source

水稻条斑病菌BLS256

X. oryzae pv. oryzicola BLS256

菲律宾

The Philippines

上海海关

Shanghai Customs District

水稻条斑病菌RS105

X. oryzae pv. oryzicola RS105

中国

China

上海海关

Shanghai Customs District

水稻条斑病菌FZ05

X. oryzae pv. oryzicola FZ05

中国

China

上海海关

Shanghai Customs District

水稻条斑病菌ACCC 05509

X. oryzae pv. oryzicola ACCC 05509

中国

China

中国农业微生物菌种保藏管理中心

Agricultural Culture Collection of China

水稻条斑病菌oxy02

X. oryzae pv. oryzicola oxy02

中国

China

中国农业科学院植物保护研究所

Institute of Plant Protection, Chinese Academy of Agricultural Sciences

水稻条斑病菌oxy04

X. oryzae pv. oryzicola oxy04

中国

China

中国农业科学院植物保护研究所

Institute of Plant Protection, Chinese Academy of Agricultural Sciences

水稻条斑病菌oxy05

X. oryzae pv. oryzicola oxy05

中国

China

中国农业科学院植物保护研究所

Institute of Plant Protection, Chinese Academy of Agricultural Sciences

水稻条斑病菌JS

X. oryzae pv. oryzicola JS

中国

China

浙江省农业科学院植物保护与微生物研究所

Institute of Plant Protection and Microbiology, Zhejiang

Academy of Agricultural Sciences

水稻条斑病菌AH

X. oryzae pv. oryzicola AH

中国

China

浙江省农业科学院植物保护与微生物研究所

Institute of Plant Protection and Microbiology, Zhejiang

Academy of Agricultural Sciences

水稻条斑病菌GX

X. oryzae pv. oryzicola GX

中国

China

浙江省农业科学院植物保护与微生物研究所

Institute of Plant Protection and Microbiology, Zhejiang

Academy of Agricultural Sciences

水稻条斑病菌YN

X. oryzae pv. oryzicola YN

中国

China

浙江省农业科学院植物保护与微生物研究所

Institute of Plant Protection and Microbiology, Zhejiang

Academy of Agricultural Sciences

水稻白叶枯病菌PXO86

X. oryzae pv. oryzae PXO86

菲律宾

The Philippines

上海海关

Shanghai Customs District

水稻白叶枯病菌PXO99A

X. oryzae pv. oryzae PXO99A

菲律宾

The Philippines

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌ScYc-b

X. oryzae pv. oryzae ScYc-b

中国

China

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌YN11

X. oryzae pv. oryzae YN11

中国

China

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌FuJ

X. oryzae pv. oryzae FuJ

中国

China

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌OS198

X. oryzae pv. oryzae OS198

中国

China

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌GD414

X. oryzae pv. oryzae GD414

中国

China

南京农业大学

Nanjing Agricultural University

水稻白叶枯病菌HEN11

X. oryzae pv. oryzae HEN11

中国

China

南京农业大学

Nanjing Agricultural University

野油菜黄单胞菌CGMCC 1.3408T

X. campestris CGMCC 1.3408T

英国

The United Kingdom

中国普通微生物菌种保藏管理中心

China General Microbiological Culture Collection Center

柑橘黄单胞菌Xac29-1

X. citri Xac29-1

中国

China

上海海关

Shanghai Customs District

花园黄单胞菌ATCC 19865

X. hortorum ATCC 19865

南斯拉夫

Yugoslavia

上海海关

Shanghai Customs District

白纹黄单胞菌FJ1

X. albilineans FJ1

中国

China

上海海关

Shanghai Customs District

地毯草黄单胞菌CCTCC AB 2018263

X. axonopodis CCTCC AB 2018263

中国

China

中国典型培养物保藏中心

China Center for Type Culture Collection

糖黄单胞菌ACCC 10416

X. sacchari ACCC 10416

中国

China

中国农业微生物菌种保藏管理中心

Agricultural Culture Collection of China

桑蒂黄单胞菌XQ1

X. sontii XQ1

中国

China

浙江大学生物技术研究所

Institute of Biotechnology, Zhejiang University

颖壳伯克霍尔德菌Os48

Burkholderia glumae Os48

中国

China

浙江大学生物技术研究所

Institute of Biotechnology, Zhejiang University

水稻嗜酸菌CGMCC 1.1728T

Acidovorax oryzae CGMCC 1.1728T

日本

Japan

中国普通微生物菌种保藏管理中心

China General Microbiological Culture Collection Center

水稻迪克菌ACCC 61554T

Dickeya oryzae ACCC 61554T

中国

China

中国农业微生物菌种保藏管理中心

Agricultural Culture Collection of China

菠萝泛菌F163

Pantoea ananatis F163

中国

China

浙江大学生物技术研究所

Institute of Biotechnology, Zhejiang University
表1  本研究用于PCR检测的细菌菌株

GenBank登录号

GenBank accession No.

氨基酸数目

Amino acid number

编码序列区

Coding sequence region

功能注释

Function annotation

ACD56807423CP000967.2:135741-137012 (－)膜蛋白 Membrane protein
ACD5720464CP000967.2:564852-565046 (－)假定蛋白 Hypothetical protein
ACD5894243CP000967.2:2379718-2379849 (－)假定蛋白 Hypothetical protein
ACD6026678CP000967.2:3787928-3788164 (+)假定蛋白 Hypothetical protein
ACD61250103CP000967.2:4828782-4829093 (－)α/β水解酶 α/β hydrolase
ACD6153146CP000967.2:5112608-5112748 (+)假定蛋白 Hypothetical protein
表2  Xanthomonas oryzae pv. oryzae有而X. oryzae pv. oryzicola没有的蛋白
图1  引物XocFhuE-F/XocFhuE-R和PCR扩增序列在 fhuE 序列中对应区域的示意图
图2  引物XocFhuE-F/XocFhuE-R和对应于黄单胞菌属 fhuE 基因的核苷酸序列黑点表示与引物相同的核苷酸；A、T、G、C表示与引物不同的核苷酸。
图3  用引物XocFhuE-F/XocFhuE-R扩增159 bp目标片段的琼脂糖凝胶电泳图
图4  用实时荧光定量PCR（A）和数字PCR（B）从基因组DNA中特异检测Xoc fhuE 假基因1：X.oryzae pv. oryzicola （Xoc） BLS256；2：Xoc RS105；3：X.oryzae pv. oryzae （Xoo） PXO99A；4：Xoo PXO86；5：Xoo ScYc-b；6：Xoo YN11；7：X. campestris CGMCC 1.3408T；8：X. citri Xac29-1；9：X. hortorum ATCC 19865；10：X. albilineans FJ1；11：B. glumae Os48；12：A. oryzae CGMCC 1.1728T；13：D. oryzae ACCC 61554T；14：P. ananatis F163；15：ddH2O。
图5  用实时荧光定量PCR（A）和数字PCR（B）从不同质量浓度的Xoc基因组DNA中检测 fhuE 假基因1：1.2×106 fg/μL DNA；2：1.2×105 fg/μL DNA；3：1.2×104 fg/μL DNA；4：1.2×103 fg/μL DNA；5：1.2×102 fg/μL DNA；6：1.2×101 fg/μL DNA；7：1.2 fg/μL DNA；8：ddH2O。
图6  用实时荧光定量PCR从菌悬液（A～B）和带菌水稻种子（C～D）中检测水稻条斑病菌A～B. 1：1.6×108 CFU/mL；2：1.6×107 CFU/mL；3：1.6×106 CFU/mL；4：1.6×105 CFU/mL；5：1.6×104 CFU/mL；6：1.6×103 CFU/mL；7：ddH2O。 C～D. 1：1.2×105 CFU/粒；2：1.2×104 CFU/粒；3：1.2×103 CFU/粒；4：1.2×102 CFU/粒；5：ddH2O。
图7  用数字PCR从菌悬液（A）和带菌水稻种子（B）中检测水稻条斑病菌
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