‘Cocktail’葡萄柚黄龙病菌检测及鉴定

doi:10.16420/j.issn.0513-353x.2021-0114

园艺学报 ›› 2022, Vol. 49 ›› Issue (3): 590-596.doi: 10.16420/j.issn.0513-353x.2021-0114

‘Cocktail’葡萄柚黄龙病菌检测及鉴定

贾瑾¹, 徐云龙¹, 周佳乐¹, 杨小芬¹, 钟永辉², 曾继吾³, 洪霓¹, 王国平¹, 彭抒昂⁴, 丁芳¹^,⁴^,^*()

¹华中农业大学植物科学技术学院,湖北省作物病虫害检测与安全控制重点实验室,武汉 430070
²广东省梅州市梅县区农业科学研究所,广东梅州 514021
³广东农业科学院果树研究所,广州 510640
⁴华中农业大学园艺植物生物学教育部重点实验室,武汉 430070

收稿日期:2021-06-22 修回日期:2021-12-23 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 丁芳 E-mail:dinfany@mail.hzau.edu.cn
基金资助:
国家重点研发计划项目(2018YFD0201500);广西科技重大专项(桂科AA18118046);国家自然科学基金项目(31872077)

Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit

JIA Jin¹, XU Yunlong¹, ZHOU Jiale¹, YANG Xiaofen¹, ZHONG Yonghui², ZENG Jiwu³, HONG Ni¹, WANG Guoping¹, PENG Shu’ang⁴, DING Fang¹^,⁴^,^*()

¹Hubei Key Laboratory of Plant Pathology,College of Plant Science and Technology,Huazhong Agricultural University,Wuhan 430070,China
²Institute of Agricultural Sciences,Meixian District,Meizhou,Guangdong 514021,China
³Institute of Fruit Trees,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China
⁴Key Laboratory of Horticultural Plant Biology,Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China

Received:2021-06-22 Revised:2021-12-23 Online:2022-03-25 Published:2022-03-25
Contact: DING Fang E-mail:dinfany@mail.hzau.edu.cn

摘要/Abstract

摘要：

采用直接组织免疫印迹（Direct tissue blot immunoassay,DTBIA）和常规PCR技术,对采自广东梅州新建‘Cocktail’葡萄柚园内表现疑似黄龙病症状的样品进行检测。DTBIA检测发现疑似样品的韧皮部存在特征性紫红色反应,与黄龙病阳性对照反应一致。PCR检测发现枝条、叶片和果实样品中均检测出了黄龙病菌特异性条带,其中果蒂、中柱和种皮内病菌含量较高。进一步测序分析发现,来自‘Cocktail’葡萄柚病样的16S rDNA和efTu基因与NCBI中的柑橘黄龙病菌亚洲种（Candidatus Liberibacter asiaticus,CLas）对应基因的序列相似性均为100%,证实‘Cocktail’葡萄柚感染了柑橘黄龙病菌亚洲菌株。

关键词: 葡萄柚, 柑橘黄龙病菌, 检测, 鉴定, DTBIA, 16S rDNA, efTu, 序列分析

Abstract:

DTBIA and regular PCR were both adopted to identify Huanglongbing（HLB）bacteria in the samples showing symptoms similar to HLB in orchards in Meizhou,Guangdong. Typical purple spots were observed in the phloem of leaf petioles and branches of cocktail grapefruit by DTBIA assay,similar as those produced in HLB positive control. Further analysis with branch,leaf,fruit peduncle,columella,seed coat,and ovary by PCR revealed positive target bands were got from all the above samples. The titer of Candidatus Liberibacter asiaticus in fruit peduncle,columella,and seed coat was relatively higher as compared to other parts. 16S rDNA and efTu gene were both cloned and sequenced. The sequence identity between isolates of CLas deposited in GenBank and the ones from‘Cocktail’grapefruit was 100%. In conclusion,the pathogen infecting‘Cocktail’grapefruit was identified as CLas.

Key words: grapefruit, Candidatus Liberibacter asiaticus, detection, identification, DTBIA, 16S rDNA, efTu, sequence analysis

中图分类号:

S666.3

贾瑾, 徐云龙, 周佳乐, 杨小芬, 钟永辉, 曾继吾, 洪霓, 王国平, 彭抒昂, 丁芳. ‘Cocktail’葡萄柚黄龙病菌检测及鉴定[J]. 园艺学报, 2022, 49(3): 590-596.

JIA Jin, XU Yunlong, ZHOU Jiale, YANG Xiaofen, ZHONG Yonghui, ZENG Jiwu, HONG Ni, WANG Guoping, PENG Shu’ang, DING Fang. Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit[J]. Acta Horticulturae Sinica, 2022, 49(3): 590-596.

图/表 4

图1 ‘Cocktail’葡萄柚疑似染黄龙病植株症状 A：田间发病症状;B：斑驳黄化型;C：均匀黄化型;D：缺素黄化型;E：果实症状。

Fig. 1 Symptoms on Cocktail grapefruit in field A. Symptomatic‘Cocktail’grapefruit trees in the field;B. Blotchy mottling;C. Yellowing;D. Nutrient deficiency;E. Symptoms on‘Cocktail’grapefruit.

图2 ‘Cocktail’葡萄柚不同组织部位（A ~ G）中黄龙病菌DTBIA 检测结果 CK+：阳性对照（叶中脉,沙田柚）;A：叶柄;B,C：叶中脉;D,E：枝条;F,G：果蒂;CK-：阴性对照（健康沙田柚,CK1-：叶中脉,CK2-：果蒂）。

Fig. 2 Identification of HLB bacteria in different parts（A-G）of‘Cocktail’grapefruit CK+：Typical purple color was observed in the mid-rib of Shatianyou（Positive control）. A：Petiole;B,C：Mid-rib;D,E：Stem;F,G：Peduncle;CK-：Healthy control（CK1-：mid-rib,CK2-：Peduncle）.

图3 ‘Cocktail’葡萄柚不同组织部位PCR检测分析 M：MarkerⅢ（TIANGEN）;CK+：阳性对照（沙田柚）;CK-：阴性对照（沙田柚）;1：枝条;2：叶片;3：果蒂;4：中柱;5：可育种子外种皮;6：可育种子内种皮;7：胚;8：不可育种子外种皮;9：不可育种子内部组织;1#,2#,3#：疑似染病植株。

Fig. 3 PCR detection of HLB bacteria in different tissues of‘Cocktail’grapefruit M：MarkerⅢ（TIANGEN）、CK+：Positive control（Shatianyou）. CK-：Negative control（Shatianyou）. 1：Branch;2：Leaf;3：Fruit peduncle;4：Middle columella;5：Fertile outer seed coat;6：Fertile inner seed coat;7：Embryo;8：Infertile outer seed coat;9：Inferior internal tissue. 1#,2#,3# were samples showing HLB-like symptoms.

图4 基于黄龙病菌16S rDNA和efTu基因串联序列的系统进化树分析 Cocktail grapefruit GdMZ-gf为本试验获得的序列。

Fig. 4 Phylogenetic analysis based on the combined 16S rDNA and efTu gene sequences Cocktail grapefruit GdMZ-gf was obtained in this study.

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‘Cocktail’葡萄柚黄龙病菌检测及鉴定

Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit

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‘Cocktail’葡萄柚黄龙病菌检测及鉴定

Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit

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