浙江省番茄溃疡病病原菌分离与鉴定

doi:10.3969/j.issn.1004-1524.20240240

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (6): 1293-1300.DOI: 10.3969/j.issn.1004-1524.20240240

浙江省番茄溃疡病病原菌分离与鉴定

刘朋飞¹^,²(), 张舒涵¹^,², 洪凯¹^,², 邵越³, 楼兵干¹^,²^,^*()

1.浙江大学生物技术研究所,浙江杭州 310058
2.农业农村部作物病虫分子生物学重点实验室,浙江杭州 310058
3.温州市植物保护与土壤肥料管理站,浙江温州 325088

收稿日期:2024-03-15 出版日期:2025-06-25 发布日期:2025-07-08
作者简介:刘朋飞(1989—),男,甘肃会宁人,学士,研究方向为植物病理学。E-mail:liupf@zju.edu.cn
通讯作者: *楼兵干,E-mail:bglou@zju.edu.cn

Isolation and identification of the pathogen causing tomato canker in Zhejiang Province of China

LIU Pengfei¹^,²(), ZHANG Shuhan¹^,², HONG Kai¹^,², SHAO Yue³, LOU Binggan¹^,²^,^*()

1. Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
2. Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture and Rural Affairs, Hangzhou 310058, China
3. The Plant Protection and Soil Fertilizer Management Station of Wenzhou, Wenzhou 325088, Zhejiang, China

Received:2024-03-15 Online:2025-06-25 Published:2025-07-08

摘要/Abstract

摘要：

番茄溃疡病是危害番茄的一种毁灭性细菌病害,在浙江省尚未见有发生的相关报道。本研究对首次在浙江发生的番茄溃疡病进行病原菌分离纯化、形态学鉴定和致病性测定,共分离到7株致病菌,菌株在523培养基上呈黄色圆形光滑菌落,菌体杆状、大小为(0.40~0.75)μm×(1.20~3.30) μm、无鞭毛,接种番茄引起叶片卷曲干枯、茎秆溃疡以及果实出现鸟眼斑。16S rRNA和6个看家基因的序列测定、比对与系统发育分析显示,7株致病菌株与番茄溃疡病病原菌密执安棒形杆菌密执安亚种Clavibacter michiganensis subsp. michiganensis高度一致。综合形态、致病性和分子鉴定结果表明,浙江温州番茄上发生的新病害为番茄溃疡病。

关键词: 番茄溃疡病, 病原菌鉴定, 密执安棒形杆菌密执安亚种

Abstract:

Tomato bacterial canker, a devastating bacterial disease affecting tomatoes, has not been previously reported in Zhejiang Province. This study focused on the first occurrence of tomato bacterial canker in Zhejiang, conducting pathogen isolation, purification, morphological identification, and pathogenicity testing. Seven pathogenic strains were isolated, exhibiting yellow circular smooth colonies on 523 medium. The bacterial cells were rod-shaped, measuring (0.40-0.75) μm×(1.20-3.30) μm, and non-flagellated. Inoculation caused leaf curling and desiccation, stem cankers, and bird’s eye spots on tomato fruits. Further sequencing, comparison, and phylogenetic analysis of 16S rRNA and six housekeeping genes revealed that all seven pathogenic strains showed high consistency with Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker. Comprehensive morphological, pathogenic, and molecular identification results demonstrated that the newly emerged disease on tomatoes in Wenzhou, Zhejiang, is tomato bacterial canker.

Key words: tomato bacterial canker, pathogen identification, Clavibacter michiganensis subsp. michiganensis

中图分类号:

S436.412

刘朋飞, 张舒涵, 洪凯, 邵越, 楼兵干. 浙江省番茄溃疡病病原菌分离与鉴定[J]. 浙江农业学报, 2025, 37(6): 1293-1300.

LIU Pengfei, ZHANG Shuhan, HONG Kai, SHAO Yue, LOU Binggan. Isolation and identification of the pathogen causing tomato canker in Zhejiang Province of China[J]. Acta Agriculturae Zhejiangensis, 2025, 37(6): 1293-1300.

图/表 6

表1 实验所用引物

Table 1 Primers used in this study

基因 Gene	引物序列 Primer sequence	片段大小 Length/ bp	参考文献 Reference
16S rRNA	F:5'-CGGTCTACAACGCCCTCAAGA-3' R:5'- TGCGTGAAGCGGAAGATGTTG-3'	1 465	[9]
atpD	F:5'-CGGTCTACAACGCCCTCAAGA-3' R:5'-TGCGTGAAGCGGAAGATGTTG-3'	697	[10]
kdpA	F:5'-GTGCAGAACTTCGTCTCGG-3' R:5'-GAGCATCATGTTGATCATCG-3'	693	[11]
ppK	F:5'-GAGAACCTCATCCAGGCCCT-3' R:5'-GAGAACCTCATCCAGGCCCT-3'	604	[10]
sdhA	F:5'-CCTGGATGTTCGTGTACC-3' R:5'-GAGGACATGGAGTTCTTCC-3'	778	[11]
recA	F:5'-TCGGCAAGGGCTCGGTCATGC-3' R:5'-GGTCGCCRTCGTASGTGTACCA-3'	830	[12]
rpoB	F:5'-CATCATCAACGGCACCGAGC-3' R:5'-AAGCCGAAGGGGTTGATGCG-3'	1 037	[12]

表2 本实验中系统发育分析所用菌株

Table 2 Strains for phylogenetic analysis in this study

种名 Species name	菌株 Strain	来源 Origin
Clavibacter michiganensis subsp. michiganensis	CN0102	浙江温州(本研究) Wenzhou, Zhejiang, China
C. m. subsp. michiganensis	NCPPB2979	匈牙利Hungary
C. m. subsp. michiganensis	ATCC4450	未知Unknow
C. m. subsp. michiganensis	7291-3	甘肃酒泉 Jiuquan, Gansu, China
C. m. subsp. michiganensis	3315-3	中国China
C. m. subsp. michiganensis	UF1	美国America
C. m. subsp. michiganensis	VL527	智利Chile
C. m. subsp. michiganensis	A5747*	美国America
C. m. subsp. michiganensis	N.P.	白俄罗斯
		Republic of Belarus
C. m. subsp. michiganensis	A4758	中国China
C. m. subsp. sepedonicus	K496	美国America
C. m. subsp. insidiosus	R1-1	美国America
C. m. subsp. nebraskensis	CNK-2	美国America
C. m. subsp. tessellarius	ATCC33566	美国America
Ralstonia solanacearum	UW251	哥伦比亚Columbia

图1 番茄溃疡病田间症状 A,发病植株叶片枯萎;B,发病植株茎秆出现溃疡;C,发病果实出现鸟眼斑。

Fig.1 Symptoms of tomato canker in the field A, Wilted leaves of the diseased plants; B, Canker stem of the diseased plants; C, Bird’s eye spot on the diseased fruit.

图2 分离菌株CN0102在烟叶上的过敏性试验和在番茄上致病性试验结果 A,接种烟草叶片上出现过敏反应(右)和对照(左);B,茎秆针刺接种番茄苗发病;C,茎秆针刺对照;D,子叶涂抹接种植株发病;E,子叶涂抹对照;F,发病植株生长受到抑制(左)和对照(右);G,发病植株茎秆出现溃疡;H,针刺接种出现鸟眼斑的果实(左)和对照(右)。

Fig.2 Result of the HR test on tobaccos and pathogenic test on tomatoes of the isolate CN0102 A, Hypersensitive response (HR) on inoculated tobacco (right) and control (left); B, Disease symptom on stabing-stem inoculated tomato seedlings; C, Control for stabing-stem inoculation; D, Disease symptom on spreading-cotyledon inoculated tomato seedlings; E, Control for spreading-cotyledon inoculation; F, Diseased plants with inhibited growth (left) and control (right); G, Canker on the diseased stem; H, Bird’s eye spot on inlculated fruit (left) and control (right).

图3 分离菌株CN0102的形态特征 A,在523培养基上菌落形态;B,透射电镜下单个菌体;C,透射电镜下成对菌体。

Fig.3 Morphological characteristics of the isolate CN0102 A, Colony morphology on 523 medium; B, Individual bacterium under transmission electron microscope; C, Binate bacteria under transmission electron microscope.

图4 十五株不同密执安棒形杆菌亚种和茄科雷尔氏菌的atpD、kdpA、ppK、recA、rpoB、sdhA基因联合构建的系统发育树

Fig.4 Phylogenetic tree of concatenated sequences of atpD, kdpA, ppK, recA, rpoB, sdhA of 15 strains belonging to different C. michiganensis subsp. and R. solanacearum

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浙江省番茄溃疡病病原菌分离与鉴定

Isolation and identification of the pathogen causing tomato canker in Zhejiang Province of China

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