Abstract

Black rot and bacterial spots threaten the cultivation of cruciferous vegetables worldwide, and the development of a method that can easily detect, identify, and distinguish their respective pathogens Xanthomonas campestris pv. campestris (Xcc) and X. campestris pv. raphani (Xcr) is required. Multiple whole-genome sequences of Xcc and Xcr were aligned to identify specific regions and subsequently design gene markers. A region present in Xcr, but absent in Xcc, was detected, which was approximately 11.5 kbp in length, sandwiched between the serine protease homolog (SPH) and nicotinate phosphoribosyltransferase gene (pncB). It contained putative cellulose synthesis-related genes, whereas Xcc only had a modified cellulose synthase gene. Designed primers were pncB_fw1 and pncB_fw2 (from the pncB gene), Xcc_rv1 and Xcc_rv2 (from the modified cellulose synthesis gene), and Xcr_rv1 and Xcr_rv2 (from the putative first and second open reading frames of the gene cluster). PCR using pncB_fw1 and Xcc_rv1, or pncB_fw2 and Xcc_rv2, amplified DNA fragments only in Xcc and X. campestris pv. incanae (Xci). Xci is the causal agent of black rot of garden stock and closely related to Xcc. PCR using pncB_fw1 and Xcr_rv1, or pncB_2 and Xcr_rv2, amplified DNA fragments only in Xcr. Multiplex PCR analysis easily distinguished Xcc and Xcr from bacterial colonies isolated on growth media and detected the pathogen in symptomatic leaves. Multiplex nested PCR detected the contamination of one seed with Xcc and/or Xcr infection from 1000 seeds. Therefore, the PCR primers designed in this study therefore helped detect and discriminate between Xcc and Xcr.

Key points

• Xanthomonas campestris pv. campestris ( Xcc ) and pv. raphani ( Xcr ) were investigated.

• Novel primers were designed following whole-genome comparison analyses.

• Multiplex PCR with new primers distinguished Xcc and Xcr simultaneously.

中文翻译：

---

Xanthomonas campestris pv的检测和鉴定。Campestris和PV。特异性引物通过多重聚合酶链反应进行萝卜

摘要

黑腐病和细菌斑病威胁着全世界十字花科蔬菜的种植，并发展了一种可以轻松检测，鉴定和区分其各自病原体Xanthomonas campestris pv的方法。油菜（XCC）和X。油菜光伏。莱菔子（XCR）是必需的。将Xcc和Xcr的多个全基因组序列进行比对，以鉴定特定区域并随后设计基因标记。存在的区域中XCR，但不存在于XCC被检测到，长度约为11.5 kbp，介于丝氨酸蛋白酶同源物（SPH）和烟酸磷酸核糖基转移酶基因（pncB）之间。它包含推定的纤维素合成相关基因，而Xcc仅具有修饰的纤维素合酶基因。设计的引物是pncB_fw1和pncB_fw2（来自pncB基因），Xcc_rv1和Xcc_rv2（来自修饰的纤维素合成基因）以及Xcr_rv1和Xcr_rv2（来自基因簇的推定的第一个和第二个开放阅读框）。PCR使用pncB_fw1和Xcc_rv1，或pncB_fw2和Xcc_rv2，仅在扩增的DNA片段XCC和X。油菜光伏。印加（Xci）。XCI是园林股票的黑腐病的致病因子和密切相关的XCC。PCR使用pncB_fw1和Xcr_rv1，或pncB_2和Xcr_rv2，仅在扩增的DNA片段XCR。多重PCR分析可以轻松地将Xcc和Xcr与生长培养基上分离出的细菌菌落区分开，并在有症状的叶子中检测出病原体。多重巢式PCR检测了1000颗种子中一颗种子受到Xcc和/或Xcr感染的污染。因此，本研究设计的PCR引物因此有助于检测和区分Xcc和Xcr。

关键点

•Xanthomonas campestris pv。 campestris （ Xcc ） 和 PV。 莱菔子 （ XCR ） 进行了调查。

•根据全基因组比较分析设计了新型引物。

•使用新引物的多重PCR可同时区分Xcc和Xcr。