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

Abstract

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 特有而 Xoo 缺失的 fhuE 部分序列设计引物，筛选获得 Xoc 特异性引物 XocFhuE-F (5′-ATCGAACGATGTCACCAGGG-3′) 和 XocFhuE-R (5′-AGAAACGTGCGGCCAGATAA-3′)。利用引物对 XocFhuE-F/XocFhuE-R 仅可从 Xoc 菌株中扩增出 159 bp 片段，结合荧光染料分别建立 SYBR Green 实时荧光定量 PCR（quantitative real-time PCR, qPCR）与 EvaGreen 微滴数字 PCR（digital PCR, dPCR）检测鉴定 Xoc 的方法。在 20 μL 反应体系中加入 1 μL 模板，qPCR 检测菌悬液中 Xoc 的检出下限为 1.6 × 10⁴ CFU/mL，检测带菌种子中 Xoc 的检出下限为 1.2 × 10³ CFU / 粒；dPCR 检测菌悬液中 Xoc 的检出下限为 1.6 × 10³ CFU/mL，检测带菌种子中 Xoc 的检出下限为 1.2 × 10² CFU / 粒。综上所述，基于 Xoc 特异性引物 XocFhuE-F/XocFhuE-R 建立的 SYBR Green qPCR 与 EvaGreen dPCR 技术，可为口岸 Xoc 检疫及水稻条斑病监测预警提供高效可靠的检测手段。

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 of these diseases. Here, we found that the fhuE gene 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 fhuE pseudogene of Xoc but absent in that of Xoo. Polymerase chain reaction (PCR) amplification yielded only a 159 bp DNA fragment from Xoc strains using the primer pair XocFhuE-F/XocFhuE-R. Based on these Xoc-specific primers, we established SYBR Green quantitative real-time PCR (qPCR) and EvaGreen droplet digital PCR (dPCR) assays for the detection and identification of Xoc. In a 20 μL reaction system containing 1 μL template, the limit of detection of qPCR was 1.6 × 10⁴ CFU/mL in bacterial suspensions and 1.2 × 10³ CFU/seed in rice seeds; the limit of detection of dPCR was 1.6 × 10³ CFU/mL in bacterial suspensions and 1.2 × 10² CFU/seed in rice seeds. In conclusion, SYBR Green qPCR and EvaGreen dPCR established with the Xoc-specific primers XocFhuE-F/XocFhuE-R are efficient tools for Xoc quarantine and field monitoring of rice bacterial leaf streak.