青花菜菌核病拮抗菌XL-09的鉴定与全基因组序列分析

doi:10.3969/j.issn.1004-1524.20250661

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (8): 1628-1635.DOI: 10.3969/j.issn.1004-1524.20250661

青花菜菌核病拮抗菌XL-09的鉴定与全基因组序列分析

韦海忠¹(), 潘丽芹¹, 赵文萱², 李亚楠², 张慧娟², 蒋明²^,^*()

¹ 台州科技职业学院农业与生物工程学院, 浙江台州 318020
² 台州学院生命科学学院, 浙江台州 318000

收稿日期:2025-10-20 出版日期:2026-08-25 发布日期:2026-09-14
作者简介:韦海忠,研究方向为植物生理生化。E-mail: hzwei910@126.com
通讯作者: *蒋明,E-mail: jiangming1973@139.com
基金资助:
台州市科技计划项目(23nya07)

Identification and whole-genome sequence analysis of antagonistic bacterium XL-09 against Sclerotinia sclerotiorum in broccoli

WEI Haizhong¹(), PAN Liqin¹, ZHAO Wenxuan², LI Yanan², ZHANG Huijuan², JIANG Ming²^,^*()

¹ College of Agriculture and Bioengineering, Taizhou Vocational College of Science and Technology, Taizhou 318020, Zhejiang, China
² School of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China

Received:2025-10-20 Published:2026-08-25 Online:2026-09-14

摘要/Abstract

摘要：

核盘菌(Sclerotinia sclerotiorum)是引起菌核病的重要病原真菌,对青花菜(Brassica oleracea var. italica)生产造成严重危害。本研究以先前从青花菜叶片分离得到的内生细菌菌株XL-09为材料,通过形态学观察、生理生化特性测定及分子鉴定,明确其分类地位;采用平板对峙法和离体叶片接种法,评估该细菌对菌核病的抗性作用;并在基因组组装基础上,分析其次生代谢基因簇的组成。结果表明,XL-09菌株在LB固体培养基上菌落呈乳白色,革兰氏染色阳性,能利用D-木糖、蔗糖和L-阿拉伯糖等碳源,接触酶、硝酸盐还原和V-P反应等均为阳性。基于gyrA基因序列构建的系统发育树显示,XL-09与枯草芽孢杆菌(Bacillus subtilis)聚为一组。综合形态学、生理生化及分子鉴定结果,将该菌株鉴定为枯草芽孢杆菌。XL-09对核盘菌生长具有显著拮抗作用,处理后菌落直径明显小于对照,菌核数量显著减少;经XL-09处理的青花菜叶片在接种核盘菌后,病斑显著小于对照。基因组组装结果显示,XL-09基因组全长为4.28 Mb,含有4 525个基因,其中蛋白质编码基因4 308个;共检测到9种次生代谢产物基因簇,涉及产孢致死因子、表面活性素和芽孢杆菌素等的合成。拮抗细菌XL-09的鉴定及全基因组序列分析,为后续抗病机理研究和生物防治应用奠定了基础。

关键词: 核盘菌, 枯草芽孢杆菌, 青花菜, 拮抗作用, 基因组

Abstract:

Sclerotinia sclerotiorum is an important pathogenic fungus causing Sclerotinia disease, which poses serious threats to the production of broccoli (Brassica oleracea var. italica). In this study, an endophytic bacterial strain XL-09 previously isolated from broccoli leaves was used as material. Its taxonomic status was determined through morphological observation, physiological and biochemical characterization, and molecular identification. The antagonistic effect of this bacterium against Sclerotinia disease was evaluated using plate confrontation and detached leaf inoculation methods. Furthermore, based on genome assembly, the composition of secondary metabolite gene clusters was analyzed. The results showed that strain XL09 formed creamy white colonies on LB solid medium, was Grampositive, and could utilize D-xylose, sucrose, and L-arabinose as carbon sources. It was positive for catalase, nitrate reduction, and Voges-Proskauer reaction. Phylogenetic analysis based on gyrA gene sequences revealed that XL09 clustered with Bacillus subtilis. Combining morphological, physiological, biochemical, and molecular identification results, the strain was identified as Bacillus subtilis. XL09 exhibited significant antagonistic activity against the growth of Sclerotinia sclerotiorum, with colony diameter significantly smaller than that of the control and the number of sclerotia markedly reduced. Moreover, broccoli leaves treated with XL09 developed significantly smaller lesions after inoculation with S. sclerotiorum compared to the control. Genome assembly revealed that the complete genome of XL09 is 4.28 Mb in length, containing 4 525 genes, of which 4 308 are protein-coding genes. A total of nine secondary metabolite gene clusters were detected, involved in the synthesis of sporulation lethal factor, surfactin, and bacillocin, among others. The identification and whole genome sequence analysis of antagonistic bacterium XL09 lay a foundation for future studies on disease resistance mechanisms and biocontrol applications.

Key words: Sclerotinia sclerotiorum, Bacillus subtilis, broccoli, antagonism, genome

中图分类号:

S436.35

韦海忠, 潘丽芹, 赵文萱, 李亚楠, 张慧娟, 蒋明. 青花菜菌核病拮抗菌XL-09的鉴定与全基因组序列分析[J]. 浙江农业学报, 2026, 38(8): 1628-1635.

WEI Haizhong, PAN Liqin, ZHAO Wenxuan, LI Yanan, ZHANG Huijuan, JIANG Ming. Identification and whole-genome sequence analysis of antagonistic bacterium XL-09 against Sclerotinia sclerotiorum in broccoli[J]. Acta Agriculturae Zhejiangensis, 2026, 38(8): 1628-1635.

图/表 6

表1 XL-09菌株的生理生化特征

Table 1 Physiological and biochemical characteristics of XL-09 strain

序号 Number	试验名称 Test name	结果 Result	序号 Number	试验名称 Test name	结果 Result
1	D-木糖D-Xylose	+	9	蔗糖Sucrose	+
2	L-阿拉伯糖L-Arabinose	+	10	柠檬酸盐Citrate	+
3	半乳糖Galactose	-	11	接触酶Catalase	+
4	淀粉Starch	+	12	硝酸盐还原Nitrate reduction	+
5	D-甘露醇D-Mannitol	+	13	明胶液化Gelatin liquefaction	+
6	果糖Fructose	+	14	酪氨酸水解Tyrosine hydrolysis	-
7	乳糖Lactose	-	15	V-P测定Voges-Proskauer test	+
8	D-葡萄糖D-Glucose	+	16	苯丙氨酸脱氨酶Phenylalanine deaminase	-

图1 基于gyrA基因序列构建的系统发育树

Fig.1 Phylogenetic tree constructed based on the sequences of gyrA genes

图2 不同处理下核盘菌的生长 A,培养5 d时的菌落直径;B,培养5 d时的对照菌落生长情况;C,培养5 d时的XL-09处理的菌落生长情况;D,培养25 d时的菌核数量。柱上无相同小写字母表示差异显著(p<0.05),图3同。

Fig.2 Growth of Sclerotinia sclerotiorum under different treatments A, Colony diameter at 5 days of culture ; B, Colony growth of the control group at 5 days of culture ; C, Colony growth of XL-09 treatment at 5 days of culture; D, Number of sclerotia at 25 days of culture. No same lowercase letters on the column indicate significant (p<0.05) difference. The same as Fig. 3.

图3 不同处理下青花菜的抗病性 A,青花菜叶片的病斑直径;B和C分别是对照和处理的表型照片。

Fig.3 Disease resistance of broccoli under different treatments A, Lesion diameter of broccoli leaves ; B and C are the phenotypic photos of control and treatment, respectively.

图4 枯草芽孢杆菌XL-09菌株的基因组圈图圈图由外到内依次为基因组坐标、基因组序列正链上的基因、基因组序列负链上的基因、基因组序列上的rRNA和tRNA、基因组序列的GC含量曲线、基因组序列的GC偏倚曲线。

Fig.4 Circos plot of the Bacillus subtilis strain XL-09 genome The circle diagram from the outside to the inside is the genome coordinate, the gene on the positive chain of the genome sequence, the gene on the negative chain of the genome sequence, the rRNA and tRNA on the genome sequence, the GC content curve of the genome sequence, and the GC bias curve of the genome sequence.

表2 XL-09的次生代谢基因簇

Table 2 Secondary metabolite gene clusters of XL-09 strain

次生代谢产物 Secondary metabolite	基因名称 Gene name	基因簇大小/kb Gene cluster size/kb
产孢致死因子 Sporulation lethal factor	skfA、skfB、skfC、ybdM	22.1
表面活性素 Surfactin	nasC、nasB、yciC、yckB、srfAA、srfAB、srfAC、srfAD、gabR_1、sfp、tcyA、bsdB、pip_2	63.4
芽孢杆菌素 Bacillocin	pbpX、tdh、miaB、pksB、pksC、pksD、pksE、acpK、pksF、pksG、pksH、pksI、pksJ、pksL、pksM_1、pksM_ 2、pksN、pksR、pksS_1、aprX	105.4
芬荠素 Fengycin	gtaB_1、accD5、crt、yngG、cfiB、fadD3、bcd、ppsE、ppsD、ppsC、ppsB、ppsA、ggt、adhT	77.0
枯草三十七肽 Sublancin	sunS、lagD、sunA、speG	20.2
杆菌素Corynebactin	pncA、yueD、mbtH、dhbF、dhbB、dhbE、dhbC、dhbA、besA、yumC	47.1
枯草杆菌肽A Subtilosin A	narG、sboA、albA、albE、albF	21.6
芽孢菌溶素 Bacilysin	bacG、bacF_2、bacD、bacC_1、rocC_2、rocA、rfbC、rmlD、rfbB、rmlA、murG_2、arnB	41.4
泰乐菌素 Tylosin	gntZ、ahpC_2、ahpF、yydH、moaA_2、fdhA	21.7

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青花菜菌核病拮抗菌XL-09的鉴定与全基因组序列分析

Identification and whole-genome sequence analysis of antagonistic bacterium XL-09 against Sclerotinia sclerotiorum in broccoli

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