Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples
Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples
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DOI：
10.1371/journal.pone.0037836
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发表时间：
2012-05-24
期刊：
影响因子：
3.7
通讯作者：
Tavares, Fernando
中科院分区：
文献类型：
--
作者：
Albuquerque, Pedro;Caridade, Cristina M. R.;Tavares, Fernando
背景资料：细菌性斑点致病性黄单胞菌（BSX）是检疫性植物病原细菌，在番茄和辣椒生产中造成严重损失。尽管对改进的植物喷雾方法和抗性品种进行了研究，但使用健康的植物材料仍然被认为是最有效的细菌斑点控制措施。因此，快速，高效的检测方法是至关重要的早期检测这些phytopathogne.Methodology：在这项工作中，我们选择和验证新的DNA标记可靠的检测BSX黄单胞菌euvesicatoria（XEU）。使用两个在线应用程序CUPID和Insignia选择Xeu特异性DNA区域。此外，为了便于选择推定的DNA标记，定制的C程序被设计用于检索由两个数据库输出的区域。通过评估染色体位置、GC含量、密码子使用和同线性分析，进一步扩展了计算机验证，以深入了解这些Xeu-specific区域的起源。设计引物对扩增这些区域，PCR验证试验表明，大多数引物允许与不同的Xeu菌株的阳性扩增。将获得的扩增子标记并用作斑点印迹测定中的探针，其允许针对12种非BSX黄单胞菌属和23种其它植物病原性细菌的集合测试探针。这些测定证实了所选DNA标记物的特异性。最后，我们设计并测试了一个双重PCR检测和反向斑点印迹平台的培养独立检测Xeu在受感染plants.Significance：本研究详细介绍了一种选择策略，能够提供大量的Xeu特异性DNA标记。如所证明的，所选择的标记物可以通过PCR和基于杂交的测定结合自动数据分析来检测受感染植物中的Xeu。此外，这项工作有助于实现更有效的基于DNA的细菌诊断方法。
Background: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens.Methodology: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants.Significance: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.