Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing
Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing
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DOI：
10.1094/pdis-12-19-2679-sc
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发表时间：
2020-06-01
期刊：
影响因子：
4.5
通讯作者：
Hartung, John S.
中科院分区：
文献类型：
--
作者：
Ding, Fang;Peng, Shu-ang;Hartung, John S.
‘Candidatus Liberibacter sp.’与最具破坏性的柑橘黄龙病(HLB)有关。在以前的工作中，我们建立了一种原位组织印迹法来检测甜橙中的‘亚洲钙细菌’(Clas)。我们通过用5%(w/v)的健康粗柠檬提取物预置抗OMP抗体来优化实验方案。这个简单的过程消除了柑橘和抗体之间的交叉反应。优化后的方法加强了多克隆抗体的应用，我们对来自世界各地的Clas进行了检测，包括来自日本、泰国、越南、巴基斯坦、沙特阿拉伯、巴西、美国的分离物，以及来自中国的一批代表那里现存多样性的菌株。该方法还被用来检测代表南非多样性的四个非洲钙细菌(Ca.L.africanus)菌株。对代表菌株相应的外膜基因进行了克隆和测序。编码序列高度保守，Clas和Claf分离株在氨基酸水平上有53.8%~55.9%的同源性。优化后的方法对柑橘不同组织韧皮部细胞中Clas和Claf的识别都是有效的，而不考虑HLB样品的地理来源。该方法简单、重复性好，满足了对HLB细菌准确、灵敏、高通量筛选的迫切需要，特别是在植物检验检疫工作中可能发挥重要作用。
'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by prein-cubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.