柑橘黄龙病菌原噬菌体的遗传多样性分析

doi:10.16420/j.issn.0513-353x.2023-0187

摘要/Abstract

摘要：

以采自广东、广西、海南、云南、四川、贵州等6个地区共300份感染柑橘黄龙病菌的DNA样本作为试验材料，通过PCR技术对黄龙病菌携带的3种类型原噬菌体基因进行特异扩增、测序，利用相关生物信息学软件进行遗传多样性分析。结果共鉴定出8种原噬菌体组合；携带单一原噬菌体类型的黄龙病菌株为6个地理种群的优势菌株，占比53.3%，其中以Type 2型为主；3类原噬菌体基因位点中A + T含量均高于G + C，表现出AT偏好特点；重组分析结果显示黄龙病菌间无重组信号，表明未发生重组事件；基因流N_m值均大于1，说明黄龙病菌在各种群中基因交流频繁；中性检验结果显示Tajima’s D、Fu and Li’s D、Fu and Li’s F值均小于0，表明黄龙病菌种群存在过历史扩张。

关键词: 柑橘, 黄龙病, 原噬菌体, 遗传多样性

Abstract:

To explore the geographical distribution and genetic variability of Candidatus Liberibacter asiaticus（CLas）harboring diverse prophage types in China，300 CLas DNA samples extracted from leaf petioles of CLas infected citrus were collected from six provinces：Guangdong，Guangxi，Hainan，Yunnan，Sichuan and Guizhou. And surveyed for Type 1，Type 2 and Type 3 prophages by specific PCR，then sequenced，and subjected to genetic diversity analysis employing relevant bioinformatics software. The results showed that the presence of eight distinct prophage combinations among the six geographically diverse populations. Notably，the prevalence of CLas strains carrying a single prophage type was dominant，accounting for 53.3% of the samples，with Type 2 prophage being the most prevalent. Analysis of the three prophage gene loci demonstrated a higher content of A + T relative to G + C，indicating an AT preference. Recombination analysis indicated an absence of recombination signals，suggesting the absence of recombination events. The N_m value of gene flow is exceeded 1，indicating frequent gene exchange among different CLas groups. The results of the neutral test，characterized by Tajima’s D，Fu and Li’s D，Fu and Li’s F values，all being less than 0，indicated a historical expansion within the CLas population.

Key words: citrus, Huanglongbing, prophage, genetic diversity

游平, 杨进, 周俊, 黄爱军, 鲍敏丽, 易龙. 柑橘黄龙病菌原噬菌体的遗传多样性分析[J]. 园艺学报, 2024, 51(4): 727-736.

YOU Ping, YANG Jin, ZHOU Jun, HUANG Aijun, BAO Minli, YI Long. Genetic Diversity Analysis of Candidatus Liberibacter Asiaticus Based on Different Types of Prophage[J]. Acta Horticulturae Sinica, 2024, 51(4): 727-736.

图/表 8

参考文献 30

[1]	Bai Ziqin. 2012. Occurrence dynamics of citrus huanglongbing in P. R. China and population differentiation of Candidatus Liberibacter asiaticus[M. D. Dissertation]. Chongqing: Southwest University. (in Chinese)
	柏自琴. 2012. 中国柑橘黄龙病发生动态及其病原菌亚洲种群分化研究[硕士论文]. 重庆: 西南大学.
[2]	Bastianel C, Garnier-Semancik M, Renaudin J, Bové J M, Eveillard S. 2005. Diversity of“Candidatus Liberibacter asiaticus”,based on the omp gene sequence. Applied and Environmental Microbiology, 71 (11):6473-6478. doi: 10.1128/AEM.71.11.6473-6478.2005 pmid: 16269671
[3]	Bové J M. 2006. Huanglongbing:a destructive,newly-emerging,century-old disease of citrus. Journal of Plant Pathology, 88 (1):7-37.
[4]	Boyd E F, Brüssow H. 2002. Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends in Microbiology, 10 (11):521-529. pmid: 12419617
[5]	Chen J, Deng X, Sun X, Jones D, Irey M, Civerolo E. 2010. Guangdong and Florida populations of‘Candidatus Liberibacter asiaticus’distinguished by a genomic locus with short tandem repeats. Phytopathology, 100 (6):567-572. doi: 10.1094/PHYTO-100-6-0567 pmid: 20465412
[6]	Chen Yanling, Zheng Zheng, Xu Meirong, Deng Xiaoling. 2019. Molecular identification and genetic diversity of“Candidatus Liberibacter asiaticus”in W. Murcott tangerine. Acta Horticulturae Sinica, 46 (6):1155-1162. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0714
	陈燕玲, 郑正, 许美容, 邓晓玲. 2019. 杂柑‘W · 默科特’黄龙病菌分子鉴定和遗传多样性分析. 园艺学报, 46 (6):1155-1162. doi: 10.16420/j.issn.0513-353x.2018-0714
[7]	Deng Xiaoling, Zheng Yongqin, Zheng Zheng, Xu Meirong. 2019. Current research on genomic analysis of“Candidatus Liberibacter spp.”. Journal of South China Agricultural University, 40 (5):137-148. (in Chinese)
	邓晓玲, 郑永钦, 郑正, 许美容. 2019. 柑橘黄龙病菌基因组学的研究进展. 华南农业大学学报, 40 (5):137-148.
[8]	Deng Xiuxin. 2022. Review and prospect of citrus breeding in China for 60 years. Acta Horticulturae Sinica, 49 (10):2063-2074. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0701
	邓秀新. 2022. 中国柑橘育种60年回顾与展望. 园艺学报, 49 (10):2063-2074. doi: 10.16420/j.issn.0513-353x.2021-0701
[9]	Duan Y, Zhou L, Hall D G, Li W, Gottwald T. 2009. Complete genome sequence of citrus Huanglongbing bacterium,‘Candidatus Liberibacter asiaticus’obtained through metagenomics. Molecular Plant Microbe Interactions, 22 (8):1011-1020.
[10]	Fortier L C, Sekulovic O. 2013. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence, 4 (5):354-365.
[11]	Fu S, Bai Z, Su H, Liu J, Hartung J S, Zhou C, Wang X. 2020. Occurrence of prophage and historical perspectives associated with the dissemination of Huanglongbing in mainland China. Plant Pathology, 69 (1):132-138.
[12]	Garcia A G, Jamielniak J A, Diniz A, Parra J. 2022. The importance of Integrated Pest Management to flatten the Huanglongbing(HLB)curve and limit its vector,the Asian citrus psyllid. Entomologia generalis, 42 (3):349-359.
[13]	Gupta K N, Baranwal V K, Haq Q. 2012. Sequence analysis and comparison of 16S rRNA,23S rRNA and 16S/23S intergenic spacer region of greening bacterium associated with yellowing disease(Huanglongbing)of Kinnow Mandarin. Indian Journal of Microbiology, 52 (1):13-21. doi: 10.1007/s12088-011-0113-6 pmid: 23450073
[14]	Huang M, Roose M L, Yu Q B, Stover E, Hall D G, Deng Z N, Gmitter J F G. 2023. Mapping of QTLs and candidate genes associated with multiple phenotypic traits for Huanglongbing tolerance in citrus. Horticultural Plant Journal, 9 (4):705-719.
[15]	Jagoueix S, Bové J M, Garnier M. 1994. The phloemlimited bacterium of greening disease of citrus is a member of the a subdivision of the Proteobacteria. International Journal of Systematic Bacteriology, 44 (3):379-386. doi: 10.1099/00207713-44-3-379 pmid: 7520729
[16]	Jagoueix S, Bové J M, Garnier M. 1996. PCR detection of the two Candidatus Liberibacter species associated with greening disease of citrus. Molecular and Cellular Probes, 10 (1):43-50. pmid: 8684375
[17]	Li Zhenxi, Pan Ruixuan, Xu Meirong, Zheng Zheng, Deng Xiaoling. 2023. Development of duplex real-time PCR assay of‘Candidatus Liberibacter asiatics’. Acta Horticulturae Sinica, 50 (1):188-196. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0502
	李镇希, 潘睿翾, 许美容, 郑正, 邓晓玲. 2023. 柑橘黄龙病菌双重实时荧光PCR检测方法的建立. 园艺学报, 50 (1):188-196. doi: 10.16420/j.issn.0513-353x.2021-0502
[18]	Liu R, Zhang P, Pu X, Xing X Q. 2011. Analysis of a prophage gene frequency revealed population variation of‘Candidatus Liberibacter asiaticus’from two citrus-growing provinces in China. Plant Disease, 95 (4):431-435.
[19]	Nei M. 1989. Molecular evolutionary genetics. Journal of Human Evolution, 18 (8):808-810.
[20]	Song Xiao-bing, Peng Ai-tian, Huang Feng, Tang Ya-fei, Cui Yi-ping, Ling Jin-feng. 2021. Establishment of RPA rapid detection method for Asian species of“Candidatus Liberibacter asiaticus”. Jiangsu Agricultural Sciences, 49 (22):137-140. (in Chinese)
	宋晓兵, 彭埃天, 黄峰, 汤亚飞, 崔一平, 凌金锋. 2021. 柑橘黄龙病亚洲种RPA快速检测方法的建立. 江苏农业科学, 49 (22):137-140.
[21]	Su Hua-nan, Wang Xue-feng, Huang Ai-jun, Li Zhong-an, Tang Ke-zhi, Zhou Chang-yong. 2014. High quality total nucleic acid extraction method from Citrus for disease detection. Acta Horticulturae Sinica, 41 (11):2342-2352. (in Chinese)
	苏华楠, 王雪峰, 黄爱军, 李中安, 唐科志, 周常勇. 2014. 高质量提取柑橘样品中病原总核酸方法的建立. 园艺学报, 41 (11):2342-2352.
[22]	Wu Liu, Bai Xiaojing, Wen Qingli, Xie Zhu, He Yongrui, Wang Lijuan, Chen Shanchun, Zou Xiuping. 2018. Early spread characteristics of Candidatus Liberibacter asiaticus in Jincheng orange(Citrus sinensis Osbeck)by leafdisc grafting. Acta Horticulturae Sinica, 45 (11):2121-2128. (in Chinese)
	吴柳, 白晓晶, 文庆利, 谢竹, 何永睿, 王丽娟, 陈善春, 邹修平. 2018. 柑橘黄龙病病原菌Las在叶圆片嫁接接种的‘锦橙’中早期扩散研究. 园艺学报, 45 (11):2121-2128. doi: 10.16420/j.issn.0513-353x.2018-0317
[23]	Yi Long, Lu Zhan-jun, Zhong Ba-lian, Lai Xiao-hua, Chen Ci-xiang. 2011. Molecular characterization of citrus huanglongbing bacteria in Gannan navel orange based on 16S rDNA sequences alignment. Journal of Fruit Science, 28 (6):1099-1103. (in Chinese)
	易龙, 卢占军, 钟八莲, 赖晓桦, 陈慈相. 2011. 基于16S rDNA序列比对分析赣南脐橙上柑橘黄龙病病原的分子特征. 果树学报, 28 (6):1099-1103.
[24]	Zhang L, Li Z Y, Bao M L, Li T, Fang F, Zheng Y Q, Liu Y X, Xu M R, Chen J C, Deng X L, Zheng Z. 2021. A novel microviridae phage (CLasMV1)from“Candidatus Liberibacter asiaticus”. Frontiers in Microbiology, 12:754245.
[25]	Zhang S, Flores-Cruz Z, Zhou L, Kang B H, Fleites L A, Gooch M D, Wulff N A, Davis M J, Duan Y P, Gabriel D W. 2011. “Ca. Liberibacter asiaticus”carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections. Molecular Plant Microbe Interactions, 24 (4):458-468.
[26]	Zheng Lin, Wang Shuai, Liu Yunuo, Du Meixia, Peng Aihong, He Yongrui, Chen Shanchun, Zou Xiuping. 2022. Gene cloning and expression analysis of NAC gene in citrus in response to Huanglongbing. Acta Horticulturae Sinica, 49 (7):1441-1457. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0553
	郑林, 王帅, 刘语诺, 杜美霞, 彭爱红, 何永睿, 陈善春, 邹修平. 2022. 柑橘响应黄龙病菌侵染的NAC基因的克隆及表达分析. 园艺学报, 49 (7):1441-1457. doi: 10.16420/j.issn.0513-353x.2021-0553
[27]	Zheng Y Q, Huang H X, Huang Z H, Deng X L, Zheng Z, Xu M R. 2021. Prophage region and short tandem repeats of“Candidatus Liberibacter asiaticus”reveal significant population structure in China. Plant Pathology, 70 (4):959-969.
[28]	Zheng Z, Bao M L, Wu F N, Horn C V, Chen J C, Deng X L. 2018a. A type 3 prophage of‘Candidatus Liberibacter asiaticus’carrying a restriction-modification system. Phytopathology, 108 (4):454-461.
[29]	Zheng Z, Chen J C, Deng X L. 2018b. Historical perspectives,management and current research of citrus Huanglongbing in Guangdong of China,where the disease has been endemic for over a hundred years. Phytopathology, 108 (11):1224-1236.
[30]	Zhou Changyong. 2018. Reconsideration on the control strategy of citrus Huanglongbing. Plant Protection, 44 (5):30-33. (in Chinese)
	周常勇. 2018. 对柑橘黄龙病防控对策的再思考. 植物保护, 44 (5):30-33.

省份 Province	采集地点 Collection location	寄主 Host	样本数量 Number of samples
海南 Hainan	澄迈县 Chengmai County	绿橙 Green Orange	11
	琼海市 Qionghai City	柠檬 Lemon	9
	昌江县 Changjiang County	金橘 Kumquat	2
	琼中县 Qiongzhong County	绿橙 Green Orange	12
	海口市 Haikou City	绿橙 Green Orange	16
云南 Yunnan	丽江市 Lijiang City	碰柑 Ponkan	12
	建水县 Jianshui County	脐橙 Navel Orange	17
	德宏州 Dehong Prefecture	茂谷柑 Murcott	3
	鹤庆县 Heqing County	茂谷柑 Murcott	3
	宾川县 Binchuan County	茂谷柑 Murcott	7
	红河市 Honghe City	茂谷柑 Murcott	4
	玉溪市 Yuxi City	沃柑 Orah Mandarin	4
四川 Sichuan	宜宾市 Yibin City	沙糖橘 Shatangju Mandarin	6
	宁南县 Ningnan County	脐橙 Navel Orange	5
	攀枝花市 Panzhihua City	沃柑 Orah Mandarin	30
	雷波县 Leibo County	脐橙 Navel Orange	9
广西 Guangxi	柳州市 Liuzhou City	马水橘 Mashui Orange	5
	河池市 Hechi City	皇帝柑 Huangdi Mandarin	5
	来宾市 Laibin City	沙糖橘 Shatangju Mandarin	5
	南宁市 Nanning City	沙糖橘 Shatangju Mandarin	5
	玉林市 Yulin City	化橘红 Pummelo Peel	5
	桂林市 Guilin City	沙糖橘 Shatangju Mandarin	6
	横县 Heng County	沃柑 Orah Mandarin	5
	梧州市 Wuzhou City	沃柑 Orah Mandarin	7
	贺州市 Hezhou City	沃柑 Orah Mandarin	5
	崇左市 Chongzuo City	沙糖橘 Shatangju Mandarin	2
广东 Guangdong	肇庆市 Zhaoqing City	沙糖橘 Shatangju Mandarin	10
	阳江市 Yangjiang City	沙糖橘 Shatangju Mandarin	11
	博罗县 Boluo County	沙糖橘 Shatangju Mandarin	19
	四会市 Sihui City	沙糖橘 Shatangju Mandarin	10
贵州 Guizhou	罗甸县^* Luodian County	脐橙 Navel Orange	50

省份 Province	采集地点 Collection location	寄主 Host	样本数量 Number of samples
海南 Hainan	澄迈县 Chengmai County	绿橙 Green Orange	11
	琼海市 Qionghai City	柠檬 Lemon	9
	昌江县 Changjiang County	金橘 Kumquat	2
	琼中县 Qiongzhong County	绿橙 Green Orange	12
	海口市 Haikou City	绿橙 Green Orange	16
云南 Yunnan	丽江市 Lijiang City	碰柑 Ponkan	12
	建水县 Jianshui County	脐橙 Navel Orange	17
	德宏州 Dehong Prefecture	茂谷柑 Murcott	3
	鹤庆县 Heqing County	茂谷柑 Murcott	3
	宾川县 Binchuan County	茂谷柑 Murcott	7
	红河市 Honghe City	茂谷柑 Murcott	4
	玉溪市 Yuxi City	沃柑 Orah Mandarin	4
四川 Sichuan	宜宾市 Yibin City	沙糖橘 Shatangju Mandarin	6
	宁南县 Ningnan County	脐橙 Navel Orange	5
	攀枝花市 Panzhihua City	沃柑 Orah Mandarin	30
	雷波县 Leibo County	脐橙 Navel Orange	9
广西 Guangxi	柳州市 Liuzhou City	马水橘 Mashui Orange	5
	河池市 Hechi City	皇帝柑 Huangdi Mandarin	5
	来宾市 Laibin City	沙糖橘 Shatangju Mandarin	5
	南宁市 Nanning City	沙糖橘 Shatangju Mandarin	5
	玉林市 Yulin City	化橘红 Pummelo Peel	5
	桂林市 Guilin City	沙糖橘 Shatangju Mandarin	6
	横县 Heng County	沃柑 Orah Mandarin	5
	梧州市 Wuzhou City	沃柑 Orah Mandarin	7
	贺州市 Hezhou City	沃柑 Orah Mandarin	5
	崇左市 Chongzuo City	沙糖橘 Shatangju Mandarin	2
广东 Guangdong	肇庆市 Zhaoqing City	沙糖橘 Shatangju Mandarin	10
	阳江市 Yangjiang City	沙糖橘 Shatangju Mandarin	11
	博罗县 Boluo County	沙糖橘 Shatangju Mandarin	19
	四会市 Sihui City	沙糖橘 Shatangju Mandarin	10
贵州 Guizhou	罗甸县^* Luodian County	脐橙 Navel Orange	50

原噬菌体类 Prophage type	基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	参考文献 Reference
Type 1	SC1_gp035	F：TGGCTCGGGTTCAGGTAAAT；R：AAGGGCGACGCATGTATTTC	60	Zheng et al.,2018a
Type 2	SC2_gp030	F：ACCCTCGCACCATCATGTTA；R：TCGTCTTGATTGGGCAGAGT	60	Zheng et al.,2018a
Type 3	P_JXGC_gp08	F：CTGATCCTTTACCATGCCGC；R：CAGCGAAACCGATCTTGAGG	60	Zheng et al.,2018a

原噬菌体类 Prophage type	基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	参考文献 Reference
Type 1	SC1_gp035	F：TGGCTCGGGTTCAGGTAAAT；R：AAGGGCGACGCATGTATTTC	60	Zheng et al.,2018a
Type 2	SC2_gp030	F：ACCCTCGCACCATCATGTTA；R：TCGTCTTGATTGGGCAGAGT	60	Zheng et al.,2018a
Type 3	P_JXGC_gp08	F：CTGATCCTTTACCATGCCGC；R：CAGCGAAACCGATCTTGAGG	60	Zheng et al.,2018a

原噬菌体类型 Prophage type	广东 Guangdong	广西 Guangxi	四川 Sichuan	贵州 Guizhou	云南 Yunnan	海南 Hainan	总数 Total
Type 1	2（4.0%）	1（2.0%）	7（14.0%）	2（4.0%）	21（42.0%）	4（8.0%）	37（12.3%）
Type 2	35（70.0%）	35（70.0%）	2（4.0%）	12（24.0%）	9（18.0%）	24（48.0%）	117（39.0%）
Type 3	2（4.0%）	2（4.0%）	1（2.0%）	0	0	1（2.0%）	6（2.0%）
Type 1 + Type 2	9（18.0%）	0	3（6.0%）	19（38.0%）	6（12.0%）	4（8.0%）	41（13.7%）
Type 1 + Type 3	0	3（6.0%）	24（48.0%）	4（6.0%）	4（8.0%）	5（10.0%）	40（13.3%）
Type 2 + Type 3	0	3（6.0%）	0	0	0	0	3（1.0%）
Type 1 + Type 2 + Type 3	0	0	8（16.0%）	13（26.0%）	0	3（6.0%）	24（8.0%）
NONE	2（4.0%）	6（12.0%）	5（10.0%）	0	10（20.0%）	9（18.0%）	32（10.7%）