一株番茄青枯病生防细菌的筛选、鉴定及其生防潜力分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0598

摘要/Abstract

摘要：

【目的】茄科罗尔斯通氏菌（Ralstonia solanacearum, Rs）引起的青枯病是番茄的主要病害。从番茄根际筛选拮抗青枯病菌且具有促生功能的菌株，探究其生理生化特性以及对番茄青枯病的防效，为进一步开发生防制剂提供理论依据。【方法】采用滤纸片法筛选拮抗菌株，通过生理生化特性、16S rRNA序列对菌株进行鉴定分析，采用盆栽试验评价其生防和促生效果，利用16S rRNA基因扩增子测序和荧光定量PCR技术探究拮抗菌株对番茄根际细菌群落的影响。【结果】筛选到拮抗番茄青枯病菌的一株优良菌株A72，经16S rRNA序列鉴定菌株A72为暹罗芽胞杆菌，该菌株具有解磷、解钾、产IAA以及分泌胞外水解酶、铁载体和形成生物膜的能力。盆栽试验表明其对番茄青枯病的防病效果为63.80%，且该菌株能够显著提高番茄植株的根长、株高、干重、鲜重和叶绿素含量。施用菌株A72显著降低了番茄根际土壤中青枯菌的密度并改变了根际细菌群落的结构和组成。【结论】菌株A72对番茄幼苗具有良好的促生防病效果。

关键词: 拮抗菌株, 番茄青枯病菌, 根际细菌群落, 暹罗芽胞杆菌, 生物防治

Abstract:

【Objective】Tomato bacterial wilt caused by Ralstonia solanacearum（Rs）is main disease of tomato. In this study, the antagonistic strain against R. solanacearum and with growth promoting was screened from tomato rhizosphere, and its physiological and biochemical characteristic and biocontrol effect were investigated, which may provide a theoretical basis for the further development of biological control agents.【Method】Filter paper slice method was employed to screen the antagonistic strain against Rs. Physiological and biochemical characteristic and 16S rRNA sequences were analyzed to identify the antagonistic strain. Pot experiments were conducted to evaluate the biocontrol and growth promoting effects of the antagonistic strain on bacterial wilt and tomato, respectively. 16S rRNA gene amplicon sequencing and fluorescence quantitative PCR were used to investigate the effect of antagonistic strain on the bacterial community in tomato rhizosphere.【Result】An antagonistic strain A72 was screened out from the tomato rhizosphere. Strain A72 was identified as Bacillus siamensis by 16S rRNA gene sequence, and it had the ability to resolve phosphorus, release potassium, produce IAA, secrete extracellular hydrolases and siderophore, and form biofilms. The pot experiments showed that its control effect against tomato bacterial wilt was 63.80%, and it significantly increased the root length, plant height, dry weight, fresh weight and chlorophyll content of tomato plants. Application of strain A72 significantly decreased the density of R. solanacearum and altered the structure and composition of bacterial community in tomato rhizosphere.【Conclusion】Strain A72 has a good growth promotion effect on tomato seedlings and may effectively control tomato bacterial wilt.

Key words: antagonistic strain, Ralstonia solanacearum, rhizosphere bacterial community, Bacillus siamensis, biocontrol

慕雪男, 吴桐, 郑子薇, 张越, 王志刚, 徐伟慧. 一株番茄青枯病生防细菌的筛选、鉴定及其生防潜力分析[J]. 生物技术通报, 2025, 41(1): 276-286.

MU Xue-nan, WU Tong, ZHENG Zi-wei, ZHANG Yue, WANG Zhi-gang, XU Wei-hui. Screening, Identification and Biocontrol Potential Analysis of an Antagonistic Strain against Ralstonia solanacearum[J]. Biotechnology Bulletin, 2025, 41(1): 276-286.

图/表 10

图1 菌株A72的筛选与系统发育树分析 A: 菌株A72对青枯菌的抑制效果；B: 菌株A72基于16S rRNA基因序列构建的系统发育树

Fig. 1 Screening of strain A72 and phylogenetic tree analysis A: Inhibitory effect of strain A72 on R. solanacearum. B: Phylogenetic tree of strain A72 based on 16S rRNA gene sequence

图2 B. siamensis A72的生理生化特性与生物膜形成能力 A：生理生化特性；B：生物膜形成能力。不同小写字母表示处理间差异显著（P<0.05），下同

Fig. 2 Physiological and biochemical characteristics and biofilm forming ability of B. siamensis A72 A: Determination of physiological and biochemical characteristics. B: Biofilm forming ability. The different lowercase letters indicate significant differences among treatments（P<0.05）. The same below

表1 B. siamensis A72的生理生化特性

Table 1 Physiological and biochemical characteristics of B. siamensis A72

试验项目 Test item	结果 Result	试验项目 Test item	结果 Result
蛋白酶 Protease	+	固氮 Nitrogen fixation	-
淀粉酶 Amylase	+	解磷 Phosphorus resolving	+
纤维素酶 Cellulase	+	解钾 Potassium releasing	+
铁载体 Siderophore	+	IAA产率 IAA production/（µg·mL^-1）	35.17±1.91

图3 不同处理中番茄幼苗接种青枯菌后的发病情况 CK1：清水+青枯病菌；CK2：无菌NB培养基+青枯病菌；CD：多菌灵+青枯病菌；A72：生防菌A72+青枯病菌

Fig. 3 Symptoms of tomato seedlings with R. solanacearum inoculation in different treatments CK1: Water + Ralstonia solanacearum; CK2: sterile NB medium + Ralstonia solanacearum; CD: carbendazim + Ralstonia solanacearum; A72: biological control strains A72 + Ralstonia solanacearum

表2 B. siamensis A72对番茄青枯病的防病效果

Table 2 Control effect of B. siamensis A72 on tomato bacterial wilt

处理 Treatment	接种青枯菌后16 d after inoculation with Rs		接种青枯菌后20 d after inoculation with Rs
处理 Treatment	病情指数Disease index	防治效果Control efficiency/%	病情指数Disease index	防治效果Control efficiency/%
CK1	22.50±2.50 a		35.00±5.00 b
CK2	25.83±1.44 a		48.33±5.77 a
CD	15.00±2.50 b	42.12±7.06 a	25.00±2.50 c	48.48±6.15 b
A72	11.67±2.89 b	55.15±8.40 a	17.50±2.50 c	63.80±2.78 a

图4 不同处理中番茄根际细菌群落组成 A：基于 Bray-Curtis 距离的细菌群落的主坐标分析（PCoA）；B：不同处理中门水平上的相对丰度（Student's t 检验, P<0.05）；C：不同处理中属水平上的相对丰度；D：不同处理中细菌属的聚类分析热图

Fig. 4 Composition of bacterial communities in the tomato rhizosphere in different treatments A: Principal coordinate analysis of bacterial communities based on Bray-Curtis distance（PCoA）. B: Relative abundance（%）of all bacterial phyla in different treatments（Student's t test, P<0.05）. C: Relative abundance（%）of bacterial genera in different treatments. D: Heat map of cluster analysis of bacterial genera in different treatments

图5 不同处理中根际细菌属水平的丰度差异和线性判别分析（LEfSe） A：不同处理中根际细菌属水平的丰度差异分析（Wilcoxon秩和检验, P<0.05）；B：不同处理中根际细菌属水平的线性判别分析（LDA≥2.5）

Fig. 5 Analysis of differential abundance and linear discriminant analysis（LEfSe）of rhizosphere bacterial genera in different treatments A: Analysis of differential abundance rhizosphere bacterial genera in different treatments（Wilcoxon Kruskal-Wallis, P<0.05）. B: Linear discriminant analysis of rhizosphere bacterial genera in different treatments（LDA≥2.5）

图6 根际细菌属与病情指数的相关性分析和根际芽胞杆菌属、茄科罗尔斯通氏菌绝对丰度 A：根据皮尔逊（Pearson）相关系数分析根际细菌属与病情指数的相关性；B：根际芽胞杆菌属的绝对丰度；C：根际茄科罗尔斯通氏菌的绝对丰度。热图对应的值为Pearson相关系数r，r>0 呈正相关，r<0 呈负相关。*，**，***分别表示在P<0.05，P<0.01，P<0.001水平存在显著性差异

Fig. 6 Analysis of correlation between rhizosphere bacterial genera and disease index, and absolute abundances of Bacillus and Ralstonia solanacearum in tomato rhizosphere A: The correlation between rhizosphere bacteria and disease index based on Pearson correlation coefficient. B: Absolute abundance of rhizosphere Bacillus. C: Absolute abundance of rhizosphere Ralstonia solanacearum. The corresponding value of the heat map is Pearson correlation coefficient r, where r > 0 is positive correlation and r < 0 is negative correlation. *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively

图7 菌株 A72 对番茄幼苗的促生效果（A）及综合能力评价（B） CSCK1：清水；CSCK2：无菌NB培养基；CSA72：菌悬液

Fig. 7 Growth promotion effect（A）and evaluation of comprehensive ability of strain A72 on tomato seedlings（bar=10 cm） CSCK1: Water; CSCK2: sterile NB medium; CSA72: bacterial suspension

图8 B. siamensis A72对土壤理化性质的影响

Fig. 8 Effects of B. siamensis A72 on soil physicochemical properties

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