柑橘溃疡病菌噬菌体的分离鉴定

doi:10.16420/j.issn.0513-353x.2020-0866

摘要/Abstract

摘要：

从中国9个地区采集柑橘溃疡病样本,并通过单菌落形态,PCR特异性引物和16S rDNA 测序对地毯草黄单胞菌柑橘致病变种进行分离鉴定,获得17株目标菌。以地毯草黄单胞菌柑橘致病变种标准菌株ACCC 03526为宿主菌,采用双层平板法从南京市污水样本中分离其噬菌体,获得1株地毯草黄单胞菌柑橘致病变种烈性噬菌体,命名为Xac-P9。通过透射电镜对噬菌体Xac-P9形态大小进行观察,结果显示Xac-P9头部直径约58 nm,属于有尾噬菌体目（Caudovirales）,短尾噬菌体科（Podoviridae）。对Xac-P9的裂解谱、最佳感染复数、一步生长曲线、热稳定性、pH稳定性、紫外线敏感性及杀菌速率等生物学特性进行测定,结果表明,Xac-P9对17株来源不同的Xac 菌株的裂解率为100%,裂解谱宽;最佳感染复数为0.001,此时所获噬菌体效价为6.6 × 10⁹ PFU · mL^-1;一步生长曲线表明：Xac-P9潜伏期为20 min,爆发时间为60 min,裂解量为132;Xac-P9在40 ~ 60 ℃条件下具有一定耐受性,在pH 3 ~ 13环境中具有活性,对紫外辐射不敏感;30 min对宿主菌ACCC 03526的杀菌率可达90%,150 min后可将宿主菌全部杀灭。

关键词: 柑橘, 溃疡病, 地毯草黄单胞菌柑橘致病变种, 噬菌体, 分离, 鉴定, 生物学特性

Abstract:

Samples were first collected from nine regions in China. A total of 17 Xanthomonas axonopodis pv. citri（Xac）strains were isolated and identified by single colony morphology,specific PCR primers and 16S rDNA sequencing step by step. Then,the standard Xac strain ACCC 03526 was selected as the host bacteria. One strain of Xac virulent phage named Xac-P9 was isolated from the sewage of Nanjing using a double-layered plating procedure. The morphological size of Xac-P9 was observed by transmission electron microscope（TEM）. The head of Xac-P9 had a diameter of about 58 nm,suggesting that it belongs to the Podoviridae,Caudovirales. The biological characteristics of Xac-P9,such as lysis spectrum,optimal multiplicity of infection（MOI）,one-step growth curve,thermal stability,pH stability,UV sensitivity and rate of bactericidal action,were determined. The lysis rate of Xac-P9 against 17 Xac strains of different origins was 100%. The highest phage titer was 6.6 × 10⁹ PFU · mL^-1 when the MOI was 0.001. The latency,burst time and burst size were 20 min,60 min and 132,respectively. Xac-P9 was tolerant at 40-60 ℃ and active in the pH range of 3 to 13. The tested phage was not sensitive to UV light;and Xac-P9 killed 90% of the host bacteria ACCC 03526 at 30 min and all host bacteria at 150 min.

Key words: citrus, citrus canker, Xanthomonas axonopodis pv. citri, bacteriophage, isolation, identification, biological properties

中图分类号:

S666

肖逍, 丁良, 丛郁, 徐旭凌, 乔欢, 何四龙, 许文建, 徐天舜. 柑橘溃疡病菌噬菌体的分离鉴定[J]. 园艺学报, 2021, 48(12): 2349-2359.

XIAO Xiao, DING Liang, CONG Yu, XU Xuling, QIAO Huan, HE Silong, XU Wenjian, XU Tianshun. Isolation and Identification of a Lytic Bacteriophage Infecting Xanthomonas axonopodis pv. citri[J]. Acta Horticulturae Sinica, 2021, 48(12): 2349-2359.

图/表 11

参考文献 17

[1]	Ahmad A A, Askora A, Kawasaki T, Fujie M, Yamada T. 2014a. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri,the causative agent of citrus canker disease. Frontiers in Microbiology, 5:1-11
[2]	Ahmad A A, Ogawa M, Kawasaki T, Fujie M, Yamada T. 2014b. Characterization of bacteriophages Cp1 and Cp2,the strain-typing agents for Xanthomonas axonopodis pv. citri. Applied and Environmental Microbiology, 80 (1):77-85.
[3]	Balogh B, Dickstein E R, Jones J B, Canteros B I. 2013. Narrow host range phages associated with citrus canker lesions in Florida and Argentina. European Journal of Plant Pathology, 135 (2):253-264. doi: 10.1007/s10658-012-0082-6 URL
[4]	Jiang Yan-hua, Wang Lian-zhu, Li Feng-ling, Zhai Yu-xiu, Yao Lin. 2015. Isolation,identification and biological properties of a lytic phage against Vibrio parahaemolyticus. Chinese Journal of Biologicals, 28 (11):1147-1152,1156. (in Chinese)
	江艳华, 王联珠, 李风铃, 翟毓秀, 姚琳. 2015. 一株副溶血性弧菌裂解性噬菌体的分离、鉴定及其生物学特性. 中国生物制品学杂志, 28 (11):1147-1152,1156.
[5]	Ma Chao, Wang Li-li, Li Xiao-yu, Xu Yong-ping. 2017. Research progress on phage control of plant bacterial diseases. Chinese Journal of Antibiotics, 42 (9):749-754. (in Chinese)
	马超, 丛聪, 王丽丽, 李晓宇, 徐永平. 2017. 噬菌体控制植物细菌性疾病的研究进展. 中国抗生素杂志, 42 (9):749-754.
[6]	Myung I S, Nam K W, Cho Y S. 2002. Cultural characteristics of Xanthomonas axonopodis pv. citri bacteriophages CP 1 from Korea. The Plant Pathology Journal, 18 (6):333-337. doi: 10.5423/PPJ.2002.18.6.333 URL
[7]	Ning Shu-xiang, Nie Li-ping, Lu Min, Ding Ming-jin. 2000. Isolation and characteristics of VP₁₀-a marine bacteriophage. Fisheries Science, 19 (2):14-16. (in Chinese)
	宁淑香, 聂丽平, 陆敏, 丁明进. 2000. VP₁₀--一株海洋噬菌体的分离及特性研究. 水产科学, 19 (2):14-16.
[8]	Qiao Huan, Xu Xu-ling, Fei Wen-bin, Huang Jie, Chen Hai, Hu Yi-lin, Cong Yu. 2019. Isolation,identification and biological properties of 3 lytic bacteriophages against AHPND-associated Vibrio parahaemolyticus. Journal of Fisheries of China, 43 (5):1378-1396. (in Chinese)
	乔欢, 徐旭凌, 费文斌, 黄杰, 陈海, 胡怿林, 丛郁. 2019. 三株AHPND致病型副溶血性弧菌烈性噬菌体的分离鉴定及其生物学特性. 水产学报, 43 (5):1378-1396.
[9]	Wang Zhong-kang, Sun Xian-yun, Xia Yu-xian, Zhou Chang-yong, Yin You-ping. 2004. Rapid detection of citrus bacterial canker disease (Xanthomonas axonopodis pv. citri)with polymerase chain reaction(PCR). Acta Phytophylacica Sinica, 34 (1):14-20. (in Chinese)
	王中康, 孙宪昀, 夏玉先, 周常勇, 殷幼平. 2004. 柑桔溃疡病菌PCR快速检验检疫技术研究. 植物病理学报, 34 (1):14-20.
[10]	Wang Zhong-kang, Tang Xian-fu, Ouyang Yi. 1990. Studied on detecting technique with the bacteriophage XCP₁ of Xanthomonas campestris pv. citri in nonsympto-matic tissues of citrus. Scientia Agricultura Sinica, 23 (2):39-44. (in Chinese)
	王中康, 唐显富, 欧阳轶. 1990. 柑桔溃疡病菌噬菌体XCP₁检验技术研究. 中国农业科学, 23 (2):39-44.
[11]	Xu Lei. 2008. Molecular identification of Xanthomonas axonopodis pv. citri and selection disease-resistant of resources[Ph. D. Dissertation]. Changsha: Hunan Agricultural University. (in Chinese)
	徐磊. 2008. 柑橘溃疡病病原菌的分子鉴定与抗病资源的筛选[博士论文]. 长沙: 湖南农业大学.
[12]	Yao Ting-shan, Zhou Chang-yong, Hu Jun-hua, Ran Chun, Li Hong-jun, Liu Hao-qiang. 2014. Screening of antagonistic Actinanyces strains against citrus canker disease and A 16 identification. Journal of Fruit Science, 31 (4):684-688. (in Chinese)
	姚廷山, 周常勇, 胡军华, 冉春, 李鸿筠, 刘浩强. 2014. 柑橘溃疡病土壤拮抗放线菌的分离及菌株A16初步鉴定. 果树学报, 31 (4):684-688.
[13]	Yao Ting-shan, Zhou Yan, Zhou Chang-yong. 2015. Research development of the differentiation and control of citrus bacterial canker disease. Acta Horticulturae Sinica, 42 (9):1699-1706. (in Chinese)
	姚廷山, 周彦, 周常勇. 2015. 柑橘溃疡病菌分化及防治研究进展. 园艺学报, 42 (9):1699-1706.
[14]	Yao Ting-shan, Zhou Yan, Zhou Chang-yong. 2016. Advances in copper resistant mechanisms and control methods of citrus canker. Acta Horticulturae Sinica, 43 (9):1711-1718. (in Chinese)
	姚廷山, 周彦, 周常勇. 2016. 应用铜制剂防治柑橘溃疡病的研究进展. 园艺学报, 43 (9):1711-1718.
[15]	Yoshikawa G, Ahmed A, Romain B M, Takeru K, Li Y Z, Miyako N, Hiroyuki O, Takashi Y. 2018. Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes. Scientific Reports, 8:1-10.
[16]	Zeng Lin, Qiu De-quan, Xie Jing-hong, Yu Ge. 2012. A study of the biochemical characteristics of the Vibrio alginolyticus bacteriophage. Journal of Guangdong Ocean University, 32 (1):24-29. (in Chinese)
	曾林, 邱德全, 谢警鸿, 于鸽. 2012. 一株溶藻弧菌噬菌体的生理特性研究. 广东海洋大学学报, 32 (1):24-29.
[17]	Zhang Ge-bi, Yang Ting-mi, Li Xi-qing, Chen Zhu-sheng. 2004. The resistance of citrus varieties to citrus canker caused by Xanthomonas citri. Acta Phytophylacica Sinica, 31 (2):111-112. (in Chinese)
	张戈壁, 阳廷密, 李喜庆, 陈竹生. 2004. 不同柑桔品种对柑桔溃疡病抗病能力的测定. 植物保护学报, 31 (2):111-112.

采样时间 Sampling time		地理来源 Geographical origin		菌株编号 Strain No.	采集部位 Sampling organ	柑橘品种 Citrus variety
2017	10-24	广西Guangxi	武鸣Wuming	Xac-1	叶片Leaf	沃柑Orah
				Xac-2	果实Fruit	沃柑Orah
	10-26		崇左Chongzuo	Xac-3	叶片Leaf	茂谷柑Murcott
				Xac-4	果实Fruit	茂谷柑Murcott
	10-29		鹿寨Luzhai	Xac-5	叶片Leaf	马水橘Mashui orange
				Xac-6	果实Fruit	马水橘Mashui orange
	11-02		河池Hechi	Xac-7	叶片Leaf	砂糖橘Shatangju
				Xac-8	果实Fruit	砂糖橘Shatangju
2018	10-17	湖南Hunan	永州Yongzhou	Xac-9	叶片Leaf	椪柑Ponkan
				Xac-10	果实Fruit	椪柑Ponkan
	10-20		麻阳Mayang	Xac-11	叶片Leaf	纽荷尔Newhall
				Xac-12	果实Fruit	纽荷尔Newhall
	10-23		靖州Jingzhou	Xac-13	叶片Leaf	血橙Blood orange
				Xac-14	果实Fruit	血橙Blood orange
	10-28	浙江Zhejiang	苍南Cangnan	Xac-15	叶片Leaf	柚 Pomelo
		浙江Zhejiang		Xac-16	果实Fruit	柚 Pomelo
	11-02	江西Jiangxi	赣州Ganzhou	Xac-17	叶片Leaf	脐橙Navel orange

采样时间 Sampling time		地理来源 Geographical origin		菌株编号 Strain No.	采集部位 Sampling organ	柑橘品种 Citrus variety
2017	10-24	广西Guangxi	武鸣Wuming	Xac-1	叶片Leaf	沃柑Orah
				Xac-2	果实Fruit	沃柑Orah
	10-26		崇左Chongzuo	Xac-3	叶片Leaf	茂谷柑Murcott
				Xac-4	果实Fruit	茂谷柑Murcott
	10-29		鹿寨Luzhai	Xac-5	叶片Leaf	马水橘Mashui orange
				Xac-6	果实Fruit	马水橘Mashui orange
	11-02		河池Hechi	Xac-7	叶片Leaf	砂糖橘Shatangju
				Xac-8	果实Fruit	砂糖橘Shatangju
2018	10-17	湖南Hunan	永州Yongzhou	Xac-9	叶片Leaf	椪柑Ponkan
				Xac-10	果实Fruit	椪柑Ponkan
	10-20		麻阳Mayang	Xac-11	叶片Leaf	纽荷尔Newhall
				Xac-12	果实Fruit	纽荷尔Newhall
	10-23		靖州Jingzhou	Xac-13	叶片Leaf	血橙Blood orange
				Xac-14	果实Fruit	血橙Blood orange
	10-28	浙江Zhejiang	苍南Cangnan	Xac-15	叶片Leaf	柚 Pomelo
		浙江Zhejiang		Xac-16	果实Fruit	柚 Pomelo
	11-02	江西Jiangxi	赣州Ganzhou	Xac-17	叶片Leaf	脐橙Navel orange

菌株名称 Strain name	菌株编号 Strain No.	裂解结果 Lysis result
地毯草黄单胞菌柑橘致病变种 Xanthomonas axonopodis pv. citri	Xac-1	+++
	Xac-2	+++
	Xac-3	+++
	Xac-4	+++
	Xac-5	+++
	Xac-6	+++
	Xac-7	+++
	Xac-8	+++
	Xac-9	+++
	Xac-10	+++
	Xac-11	+++
	Xac-12	+++
	Xac-13	+++
	Xac-14	+++
	Xac-15	+++
	Xac-16	+++
	Xac-17	+++
大肠埃希菌Escherichia coli	GIM 1.573	-
枯草芽孢杆菌Bacillus subtilis	GIM 1.784	-
短小芽孢杆菌Bacillus pumilus	GIM 1.801	-
苏云金芽孢杆菌Bacillus thuringiensis	GIM 1.150	-
棕色固氮菌Azotobacter vinelandii	GIM 1.802	-

菌株名称 Strain name	菌株编号 Strain No.	裂解结果 Lysis result
地毯草黄单胞菌柑橘致病变种 Xanthomonas axonopodis pv. citri	Xac-1	+++
	Xac-2	+++
	Xac-3	+++
	Xac-4	+++
	Xac-5	+++
	Xac-6	+++
	Xac-7	+++
	Xac-8	+++
	Xac-9	+++
	Xac-10	+++
	Xac-11	+++
	Xac-12	+++
	Xac-13	+++
	Xac-14	+++
	Xac-15	+++
	Xac-16	+++
	Xac-17	+++
大肠埃希菌Escherichia coli	GIM 1.573	-
枯草芽孢杆菌Bacillus subtilis	GIM 1.784	-
短小芽孢杆菌Bacillus pumilus	GIM 1.801	-
苏云金芽孢杆菌Bacillus thuringiensis	GIM 1.150	-
棕色固氮菌Azotobacter vinelandii	GIM 1.802	-

感染复数 MOI	Xac-P9 噬菌体数（PFU） Number of Xac-P9 phages	宿主菌数（CFU） Number of bacteria	Xac-P9效价（PFU · mL^-1） Titer of Xac-P9
1 000	1.0 × 10⁹	1.0 × 10⁶	1.2 × 10⁶
100	1.0 × 10⁸	1.0 × 10⁶	3.8 × 10⁶
10	1.0 × 10⁷	1.0 × 10⁶	1.2 × 10⁷
1	1.0 × 10⁶	1.0 × 10⁶	3.9 × 10⁸
0.1	1.0 × 10⁵	1.0 × 10⁶	4.5 × 10⁸
0.01	1.0 × 10⁴	1.0 × 10⁶	7.8 × 10⁸
0.001	1.0 × 10³	1.0 × 10⁶	6.6 × 10⁹
0.0001	1.0 × 10²	1.0 × 10⁶	4.1 × 10⁹