Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit

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

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

• Research Notes • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S666.3

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.

Figures/Tables 4

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.

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）.

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.

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

References 12

[1]	Deng X, Chen J, Feng Z, Shan Z, Guo H, Zhu J, Li H, Civerolo E L. 2008. Identification and characterization of the Huanglongbing bacterium in pummelo from multiple locations in Guangdong,P. R. China. Plant Disease, 92(4):513-518. doi: 10.1094/PDIS-92-4-0513 pmid: 30769653
[2]	Ding F, Duan Y P, Paul C, Brlansky R H, Hartung J S. 2015. Localization and distribution of‘Candidatus Liberibacter asiaticus’in citrus and periwinkle by direct tissue blot immuno assay with an Anti-OmpA polyclonal antibody. PLoS ONE, 10(5):e0123939.
[3]	Ding F, Paul C, Brlansky R H, Hartung J S. 2017. Immune tissue print and immune capture-PCR for diagnosis and detection of Candidatus Liberibacter Asiaticus. Sci Rep, 7:46467. doi: 10.1038/srep46467 URL
[4]	Ding Fang, Hong Ni, Zhong Yun, Yi Gan-jun, Wang Guo-ping. 2008. Studies on 16S rDNA sequence of citrus Huanglongbing bacteria in China. Acta Horticulturae Sinica, 35(5):649-654. (in Chinese)
	丁芳, 洪霓, 钟云, 易干军, 王国平. 2008. 中国柑橘黄龙病病原16S rDNA序列研究. 园艺学报, 35(5):649-654.
[5]	Jagoueix S, Bove J M, Garnier M. 1994. The phloem limited bacterium of greening disease of citrus is a member of the α-subdivision of the proteobacteria. International Journal of Systematic Bacteriology, 44(3):379-386. pmid: 7520729
[6]	Liu Chun-rong, Wu Xue-zhen, Wang Deng-liang, Sun Jian-cheng, Zheng Xue-liang, Cheng Hui-lin. 2020. Effect of different harvest time on fruit quality of grapefruit. Zhejiang Citrus, 37(4):28-31. (in Chinese)
	刘春荣, 吴雪珍, 王登亮, 孙建城, 郑雪良, 程慧林. 2020. 不同采收期对鸡尾葡萄柚果实品质的影响. 浙江柑橘, 37(4):28-31.
[7]	Pan R X, Chen Y L, Deng X L, Xu M R. 2021. Digital gene expression analysis of Huanglongbing affected mandarins(Citrus reticulata Blanco)in response to thermotherapy. Horticultural Plant Journal, 7(1):1-12. doi: 10.1016/j.hpj.2020.12.004 URL
[8]	Rao Xiao-zhen, Zhong Jin-liang, Wen Qing-ying, Zhang Xiong-ji, Du Xiao-zhen, Liu Rui. 2011. Preliminary report on the application of grapefruit germplasm resources in Meizhou. China Seed Industry, 3(40):65-66. (in Chinese)
	饶小珍, 钟进良, 温清英, 张雄基, 杜小珍, 刘蕊. 2011. 梅州市柚类种植资源的应用初报. 中国种业, 3(40):65-66.
[9]	Ren Jian-jie, Ye Yan-qing, Shao Li-qin, Shen Wei-long. 2020. Preliminary report on introduction of grapefruit in Shangyu,Zhejiang Province. South China Fruits, 49(5):45-46. (in Chinese)
	任建杰, 叶炎卿, 邵利琴, 沈伟龙. 2020. 鸡尾葡萄柚在浙江上虞引种试验初报. 中国南方果树, 49(5):45-46.
[10]	Shan Zhen-ju, Feng Zhen, Zhou Gen, Deng Xiao-ling. 2008. Cloning and sequence analysis of 16S rDNA fragment of pathogen of citrus HLB in Five Southern Provinces. Journal of South China Agricultural University, 29(2):25-29. (in Chinese)
	单振菊, 冯震, 周根, 邓晓玲. 2008. 南方五省区柑桔黄龙病病原16S rDNA片段的克隆与序列分析. 华南农业大学学报, 29(2):25-29.
[11]	Xia Rui, Xu Jing, Zeng Ji-wu, Zhou Bi-rong, Hung Yong-hong, Yi Gan-jun. 2009. Current status of pomelo production and trade and domestic research. Guangdong Agricultural Sciences, 7(96):328-331. (in Chinese)
	夏瑞, 徐婧, 曾继吾, 周碧容, 黄永红, 易干军. 2009. 柚类生产贸易现状及国内研究概况. 广东农业科学, 7(96):328-331.
[12]	Xie Z, Zhao K, Long J H, Zhen L, Zou X P, Chen S C. 2021. Comparative analysis of Wanjincheng orange leaf and root responses to‘Candidatus liberibacter asiaticus’ infection using leaf-disc grafting. Horticultural Plant Journal, 7(5):401-410. doi: 10.1016/j.hpj.2021.01.007 URL

[1]	WANG Rui, HONG Wenjuan, LUO Hua, ZHAO Lina, CHEN Ying, and WANG Jun, . Construction of SSR Fingerprints of Pomegranate Cultivars and Male Parent Identification of Hybrids [J]. Acta Horticulturae Sinica, 2023, 50(2): 265-278.
[2]	REN Hailong, XU Donglin, ZHANG Jing, ZOU Jiwen, LI Guangguang, ZHOU Xianyu, XIAO Wanyu, and SUN Yijia. Establishment of SNP Fingerprinting and Identification of Chinese Flowering Cabbage Varieties Based on KASP Genotyping [J]. Acta Horticulturae Sinica, 2023, 50(2): 307-318.
[3]	LIANG Jiali, WU Qisong, CHEN Guangquan, ZHANG Rong, XU Chunxiang, and FENG Shujie, . Identification of the Neopestalotiopsis musae Pathogen of Banana Leaf Spot Disease [J]. Acta Horticulturae Sinica, 2023, 50(2): 410-420.
[4]	ZHAI Hanhan, ZHAI Yujie, TIAN Yi, ZHANG Ye, YANG Li, WEN Zhiliang, CHEN Haijiang. Genome-wide Identification of Peach SAUR Gene Family and Characterization of PpSAUR5 Gene [J]. Acta Horticulturae Sinica, 2023, 50(1): 1-14.
[5]	JIANG Jingdong, WEI Zhuangmin, WANG Nan, ZHU Chenqiao, YE Junli, XIE Zongzhou, DENG Xiuxin, CHAI Lijun. Exploitation and Identification of Tetraploid Resources of Hongkong Kumquat（Fortunella hindsii） [J]. Acta Horticulturae Sinica, 2023, 50(1): 27-35.
[6]	QIU Ziwen, LIU Linmin, LIN Yongsheng, LIN Xiaojie, LI Yongyu, WU Shaohua, YANG Chao. Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata [J]. Acta Horticulturae Sinica, 2022, 49(8): 1747-1760.
[7]	CHEN Lilang, YANG Tianzhang, CAI Ruping, LIN Xiaoman, DENG Nankang, CHE Haiyan, LIN Yating, KONG Xiangyi. Molecular Detection and Identification of Viruses from Passiflora edulis in Hainan [J]. Acta Horticulturae Sinica, 2022, 49(8): 1785-1794.
[8]	LIU Peng, LI Qin, ZHANG Weirui, HE Sheqi, ZHANG Suping, MA Xiaoxu, YUAN Wangjun. Identification,Biological Characteristics and Fungicide Sensitivity of the Pathogen Causing Brown Spot Disease on Forsythia [J]. Acta Horticulturae Sinica, 2022, 49(8): 1805-1814.
[9]	XIA Yan, HUANG Song, WU Xueli, LIU Yiqi, WANG Miaomiao, SONG Chunhui, BAI Tuanhui, SONG Shangwei, PANG Hongguang, JIAO Jian, ZHENG Xianbo. Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology [J]. Acta Horticulturae Sinica, 2022, 49(7): 1415-1428.
[10]	XIAO Xuechen, LIU Mengyu, JIANG Mengqi, CHEN Yan, XUE Xiaodong, ZHOU Chengzhe, WU Xingjian, WU Junnan, GUO Yinsheng, YEH Kaiwen, LAI Zhongxiong, LIN Yuling. Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan [J]. Acta Horticulturae Sinica, 2022, 49(5): 1031-1046.
[11]	GONG Mingxia, ZHAO Hu, WANG Meng, WU Xing, ZHAO Zengjing, HE Zhi, HUANG Jinmei, MENG Shengde, WANG Risheng. Identification of Viruses Infecting Peppers in Guangxi by Small RNA Deep Sequencing and RT-PCR [J]. Acta Horticulturae Sinica, 2022, 49(5): 1060-1072.
[12]	GONG Changyi, LIU Jiaojiao, DENG Qiang, ZHANG Lixin. Identification and Pathogenicity of Colletotrichum Species Causing Anthracnose on Camellia sinensis [J]. Acta Horticulturae Sinica, 2022, 49(5): 1092-1101.
[13]	PAN Xinfeng, YE Fangting, MAO Zhijun, LI Zhaowei, FAN Kai. Genomic Identification and Molecular Evolution of the WRKY Family in Nymphaea colorata [J]. Acta Horticulturae Sinica, 2022, 49(5): 1121-1135.
[14]	YANG Yi, LI Tingyao, LI Guojing, CHEN Hancai, SHEN Zhuo, ZHOU Xuan, WU Zengxiang, WU Xinyi, ZHANG Yan. Development and Application of Insertion-Deletion（InDel）Markers in Asparagus Bean Based on Whole Genome Re-sequencing Data [J]. Acta Horticulturae Sinica, 2022, 49(4): 778-790.
[15]	WANG Ying, AI Penghui, LI Shuailei, KANG Dongru, LI Zhongai, WANG Zicheng. Identification and Expression Analysis of Genes Related to DNA Methylation in Chrysanthemum × morifolium and C. nankingense [J]. Acta Horticulturae Sinica, 2022, 49(4): 827-840.

Detection and Identification of Huanglongbing Bacteria in‘Cocktail’Grapefruit

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 4

References 12

Related Articles 15

Recommended Articles

Metrics

Comments