烟草疫霉拮抗菌XC-29的分离鉴定及其全基因组序列分析

doi:10.19586/j.2095-2341.2024.0086

生物技术进展 ›› 2024, Vol. 14 ›› Issue (6): 1004-1015.DOI: 10.19586/j.2095-2341.2024.0086

烟草疫霉拮抗菌XC-29的分离鉴定及其全基因组序列分析

汪苏洁¹(), 顾梦丽¹, 陶界锰¹^,², 童治军³, 郭俊佳¹, 金静静¹^,², 徐梦晓¹, 孟利军¹, 张剑锋¹^,², 曹培健¹^,², 卢鹏¹^,²()

^1.中国烟草总公司郑州烟草研究院，郑州 450001
^2.北京生命科技研究院，北京 102200
^3.云南省烟草农业科学研究院，昆明 650106

收稿日期:2024-04-17 接受日期:2024-06-21 出版日期:2024-11-25 发布日期:2024-12-27
通讯作者: 卢鹏
作者简介:汪苏洁 E-mail: wangsujie951230@163.com；
基金资助:
河南省科技攻关项目(212102110257);中国烟草总公司重点研发项目〔110202102050;110202201001(JY-01）〕;郑州烟草研究院省席科学家项目(902023CK0890)

Isolation, Identification and Whole-genome Sequence Analysis of Phytophthora nicotianae Antagonistic Bacteria XC-29

Sujie WANG¹(), Mengli GU¹, Jiemeng TAO¹^,², Zhijun TONG³, Junjia GUO¹, Jingjing JIN¹^,², Mengxiao XU¹, Lijun MENG¹, Jianfeng ZHANG¹^,², Peijian CAO¹^,², Peng LU¹^,²()

^1.Zhengzhou Tobacco Research Institute of China National Tobacco Corporation，Zhengzhou 450001，China
^2.Beijing Life Science Academy，Beijing 102200，China
^3.Yunan Academy of Tobacco Agricultural Sciences，Kunming 650106，China

Received:2024-04-17 Accepted:2024-06-21 Online:2024-11-25 Published:2024-12-27
Contact: Peng LU

摘要/Abstract

摘要：

烟草疫霉（Phytophthora nicotianae）是一种可引发烟草黑胫病的土传卵菌，对烟草生产造成极大危害。为了应对这一问题，筛选出一株对烟草疫霉菌具有较强生防功能的菌株XC-29，通过解析基因组信息，挖掘其拮抗代谢产物及拮抗基因。采用平板对峙法和盆栽试验鉴定生防菌XC-29的抑菌活性和防治效果；通过形态观察和16S rRNA扩增子测序技术准确鉴定了XC-29菌株；利用全基因组测序、转录组测序探究菌株拮抗机制。结果表明，根际土分离的拮抗菌株XC-29鉴定为沙福芽孢杆菌（Bacillus safensis），其全基因组测序预测出136种碳水化合物活性酶和11个编码次生代谢产物合成相关的基因簇，其中6个基因簇已被确认为抗菌物质合成簇，分别编码地衣素、双效菌素、schizokinen、丰原素、杆菌溶素和嗜铁素；转录组测序结果进一步确定菌株基因具有编码相应抑菌物质的能力。综上，沙福芽孢杆菌XC-29对烟草疫霉菌具有较强的拮抗作用，全基因组和转录组分析初步揭示了沙福芽孢杆菌XC-29的抑病机理，为进一步探究拮抗菌的抑菌机制和生物防控提供理论依据。

关键词: 烟草黑胫病, 沙福芽孢杆菌, 全基因组测序, 次级代谢产物

Abstract:

Phytophthora nicotianae is a soil-borne oomycete that can cause black shank disease of tobacco， which is very harmful to tobacco production. A strain XC-29 with strong biocontrol function against P.nicotianae was screened， and its antagonistic metabolites and genes were mined by analyzing its genomic information. The antibacterial activity and control effect of biocontrol bacterium XC-29 were verified by the plate confrontation method and pot test. The strain was identified by morphological observation and 16S rRNA amplicon sequencing. The antagonistic mechanism of strains was verified the disease prevention effect of the strain. The strains were identified by exploration by whole genome and transcriptomic sequencing. The results showed that the antagonistic strain XC-29 isolated from rhizosphere soil was identified as Bacillus safensis. Its whole genome sequencing predicted 136 carbohydrate-active enzymes and 11 gene clusters encoding the synthesis of secondary metabolites， of which six were identified as the synthesis clusters of antimicrobial substances， encoding lichenysin， zwittermicin， schizokinen， fengycin， bacilysin and bacilibactin. The results of transcriptome sequencing further confirmed that the strain genes could encode corresponding antibacterial substances. In conclusion， Bacillus safensis XC-29 has a strong antagonistic effect on Phytophthora nicotianae. The whole genome and transcriptome analysis preliminarily revealed the disease inhibition mechanism of Bacillus safensis XC-29， which provides a theoretical basis for further exploration of the antibacterial mechanism and biological prevention and control of antagonistic bacteria.

Key words: tobacco black shank, Bacillus safensis, whole genome sequencing, secondary metabolites

中图分类号:

汪苏洁, 顾梦丽, 陶界锰, 童治军, 郭俊佳, 金静静, 徐梦晓, 孟利军, 张剑锋, 曹培健, 卢鹏. 烟草疫霉拮抗菌XC-29的分离鉴定及其全基因组序列分析[J]. 生物技术进展, 2024, 14(6): 1004-1015.

Sujie WANG, Mengli GU, Jiemeng TAO, Zhijun TONG, Junjia GUO, Jingjing JIN, Mengxiao XU, Lijun MENG, Jianfeng ZHANG, Peijian CAO, Peng LU. Isolation, Identification and Whole-genome Sequence Analysis of Phytophthora nicotianae Antagonistic Bacteria XC-29[J]. Current Biotechnology, 2024, 14(6): 1004-1015.

图/表 13

图1 生防菌XC-29的生物学特性研究A：生防菌XC-29的生长曲线；B：生防菌XC-29的抑菌率；C：生防菌XC-29的抑菌图谱

Fig. 1 Study on the biological characteristics of biocontrol bacteria XC-29

图2 生防菌XC-29对对烟草黑胫病的盆栽防效注：T1—不处理；T2—生防菌处理；T3—生防菌+烟草疫霉菌谷处理；T4—烟草疫霉菌谷处理。

Fig. 2 The control effects of biocontrol bacteria XC-29 against tobacco black shank disease in pot-control

图3 生防菌XC-29形态

Fig. 3 Colony morphology of biocontrol bacteria XC-29

图4 生防菌XC-29及其相似细菌的16S rRNA基因序列构建系统进化关系及ANI值分析A：基于16S rRNA基因序列的生防菌XC-29及其相似细菌系统进化树；括号内表示为各菌株的GenBank登录号；“T”表示模式菌株；分支上的数值代表支持率（此处只显示支持率>90%的分支）；标尺表示2%的序列进化差异；B：生防菌XC-29与其相似细菌的ANI值（%）比较；图中展示的数字表示核苷酸一致性（ANI）值，单位为百分比（%）；图中颜色越红表示ANI值越高。图中涉及的菌种包括：Bacillus safensis—沙福芽孢杆菌；Bacillus pumilus—短小芽孢杆菌；Bacillus australimaris—南海芽胞杆菌；Bacillus zhangzhouensis—漳州芽孢杆菌；Bacillus xiamenensis—厦门芽孢杆菌；Bacillus aerius—空气芽孢杆菌；Bacillus altitudinis—高地芽孢杆菌；Bacillus stratosphericus—同温层芽孢杆菌；Bacillus atrophaeus—萎缩芽孢杆菌；Bacillus subtilis subsp. subtilis—枯草芽孢杆菌的一个亚种。

Fig. 4 Phylogenetic relationship and ANI value analysis based on the 16S rRNA gene sequences of biocontrol bacteria XC-29 and its similar bacteria

表1 生防菌XC-29全基因组序列分析

Table 1 Whole genome sequence analysis of biocontrol strain XC-29

类型	特征	数值
基因组	染色体个数	1
基因组	基因组序列总长度/bp	3 719 058
编码蛋白基因	基因数	3 870
	基因总长度/bp	3 310 209
	基因平均长度/bp	855
	基因序列中GC含量/%	42.24
基因间区	基因间区总长度/bp	408 849
基因间区	基因间区GC含量/%	37.87
非编码	tRNA拷贝数	81
	5S rRNA拷贝数	8
	16S rRNA拷贝数	8
	23S rRNA拷贝数	8
	sRNA拷贝数	7

表2 生防菌XC-29基因功能注释数据库分布情况

Table 2 Database distribution of gene functional annotation from the biocontrol bacterium XC-29

类型	基因数	占总基因百分比
COG	2 896	74.83%
GO	2 708	69.97%
KEGG	3 652	94.37%
CAZy	136	3.51%

图5 生防菌XC-29基因组COG功能注释图注：A—RNA加工修饰；C—能量生成和转换；D—细胞周期控制、细胞分裂和染色体分裂；E—氨基酸转运代谢；F—核苷酸转运和代谢；G—碳水化合物转运代谢；H—辅酶转运和代谢；I—脂质转运代谢；J—翻译、核糖体结构和生物合成；K—转录；L—复制，重组和修复；M—细胞壁/膜/被膜的生物合成；N—细胞运动；O—翻译后修饰，蛋白质折叠和伴侣蛋白；P—无机离子转运代谢；Q—次级代谢物生物合成、转运和代谢；R—主要功能预测；S—未知功能；T—信号转导机制；U—胞内转运、分泌和小泡运输；V—抵御机制；W—胞外结构；X—动员组—噬菌体原、转座子；Z—细胞骨架。

Fig. 5 COG function annotation of biocontrol bacterium XC-29 genome

图6 生防菌XC-29基因组GO功能注释图

Fig. 6 GO function annotation of biocontrol bacterium XC-29 genome

图7 生防菌XC-29基因组KEGG通路富集分析

Fig. 7 KEGG pathway enrichment analysis of biocontrol bacterium XC-29 genome

表3 生防菌XC-29基因组KEGG主要代谢通路分析

Table 3 Main metabolic pathways of the biocontrol bacterium XC-29 from KEGG

通路编号	类目	基因数（>40）
Map01100	代谢途径	566
Map01110	次生代谢物的生物合成	276
Map01130	抗生素的生物合成	211
Map01120	不同环境下的微生物代谢	170
Map02010	ABC转运蛋白	138
Map01230	氨基酸的生物合成	130
Map02020	双组分调节系统	111
Map01200	碳代谢	98
Map02024	群体效应	70
Map00230	嘌呤代谢	59
Map03010	核糖体	52
Map00240	嘧啶代谢	45
Map00010	糖酵解/糖异生	44
Map00620	丙酮酸代谢	44
Map00270	半胱氨酸与蛋氨酸代谢	40

表4 生防菌XC-29基因组CAZy数据库功能注释

Table 4 CAZy database function annotation of biocontrol bacterium XC-29 genome

类型	家族成员	基因数
AA	AA1	1
CBM	CBM12、CBM13、CBM48、CBM50	35
CE	CE0、CE12、CE14、CE4、CE7、CE8、CE9	18
GH	GH0、GH1、GH3、GH4、GH5、GH9、GH10、GH11、GH13、GH16、GH18、GH23、GH28、GH30、GH32、GH33、GH38、GH42、GH43、GH48、GH51、GH53、GH73、GH101、GH105、GH126、GH170、GH171	55
GT	GT0、GT1、GT4、GT9、GT26、GT28、GT30、GT51、GT58	28
PL	PL1、PL9	2

表5 生防菌XC-29次级代谢产物预测结果

Table 5 The secondary metabolite ofbiocontrol bacterium XC-29 predicted by antiSMASH

基因簇	次级代谢产物类型	基因数量	相似基因簇	相似度
簇1	非核糖体肽合成酶	43	地衣素	BGC0000381（92%）
簇2	非核糖体肽合成酶、Ⅰ型聚酮类化合物	48	双效菌素	BGC0001059（18%）
簇3	包含Rev响应元件	24	铁载体二异羟肟酸酯	-
簇4	萜类化合物、镍-铁载体	27	-	BGC0002683（60%）
簇5	β-内酯	24	丰原素	BGC0001095（53%）
簇6	萜类化合物、镍-铁载体	21	-	-
簇7	Ⅲ型聚酮类化合物	47	-	-
簇8	核糖体合成和翻译后修饰肽类似物	15	-	-
簇9	β-内酯	33	-	-
簇10	其他	46	杆菌溶素	BGC0001184（85%）
簇11	非核糖体肽-载体、非核糖体肽合成酶	39	嗜铁素bacilibactin（E/F）	BGC0002695（80%）
			嗜铁素bacilibactin	BGC0000309（100%）
			嗜铁素paenibactin	BGC0000401（100%）
			嗜铁素bacilibactin	BGC0001185（100%）

图8 生防菌XC-29编码细胞壁降解酶和次生代谢簇基因表达量A：细胞壁降解酶的基因表达量；B：次生代谢簇1的基因表达量；C：次生代谢簇5的基因表达量；D：次生代谢簇10的基因表达量；E：次生代谢簇11的基因表达量

Fig. 8 The gene expression levels of cell wall-degrading enzymes and secondary metabolite cluster genes in biocontrol bacteria XC-29

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烟草疫霉拮抗菌XC-29的分离鉴定及其全基因组序列分析

Isolation, Identification and Whole-genome Sequence Analysis of Phytophthora nicotianae Antagonistic Bacteria XC-29

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