姜瘟病生防铜绿假单胞菌的筛选和生防物质鉴定

Screening of Pseudomonas aeruginosa strains for biocontrol of bacterial wilt in ginger and identification of biocontrol substances

摘要:

背景 姜是重要经济作物，姜瘟病是影响湖南省生姜种植产业的重要病害。目的 为防控姜瘟病，从姜秆流出液中筛选铜绿假单胞菌(Pseudomonas aeruginosa)并分析细菌的生防物质。方法 提取姜瘟病姜块微生物DNA进行微生物组测序分析，平板法培养病原菌；平板法筛选姜秆流出液中铜绿假单胞菌，通过溴化十六烷基三甲胺琼脂培养基培养、42 ℃培养、氧化酶产生、绿脓菌素检测和16S rRNA基因测序鉴定菌株，平板拮抗和盆栽试验测定菌株生防效果，采用液相色谱-质谱联用仪(liquid chromatography-mass spectrometry, LC-MS)测定菌株次级代谢产物。结果 通过微生物组比对发现，发病姜块中青枯雷尔氏菌(Ralstonia solanacearum)相对丰度显著增加，从病姜中成功分离得到青枯雷尔氏菌；从姜秆流出液中筛选得到蓝绿色细菌SX6和ZJ10，经生化特性检测发现其可在溴化十六烷基三甲铵选择性培养基、42 ℃条件下生长，可产生氧化酶和绿脓菌素；平板拮抗试验显示菌株SX6和ZJ10对姜瘟病青枯雷尔氏菌抑制率为124.87%和63.08%，盆栽28 d保护率分别为42.85%和28.57%；LC-MS分析显示菌株SX6和ZJ10主要次级代谢产物为绿脓菌素，绿脓菌素粗提物对青枯雷尔氏菌抑制率分别为28.76%和25.45%。结论 湖南省郴州市汝城县小黄姜姜瘟病病原为青枯雷尔氏菌，从姜秆流出液成功获得生防铜绿假单胞菌SX6和ZJ10，绿脓菌素可能是主要拮抗物质。

Abstract:

Background Ginger is an important economic crop, and bacterial wilt is a major disease affecting ginger cultivation in Hunan province.Objective To screen Pseudomonas aeruginosa strains from ginger stem exudates and preliminarily investigate their biocontrol metabolites.Methods Microbial DNA was extracted from ginger rhizomes infected with bacterial wilt for microbiome sequencing analysis, and pathogens were cultured via the plate method. Pseudomonas aeruginosa strains were screened from ginger stem exudates by plate method, and the strains were verified by the growth experiment on cetrimide agar medium, growth test at 42 ℃, detection of oxidase and pyocyanin production, and 16S rRNA gene sequencing. The biocontrol efficacy of the strains was determined through the plate antagonism assay and pot experiments, and the secondary metabolites of the strains were analyzed by liquid chromatography-mass spectrometry (LC-MS).Results Microbiome comparison revealed that the relative abundance of Ralstonia solanacearum significantly increased in diseased ginger rhizomes, and R. solanacearum was successfully isolated from the diseased ginger. Two blue-green bacterial strains, SX6 and ZJ10, were isolated from ginger stem exudates. Biochemical characterization showed that the two strains could grow on the cetrimide agar medium and at 42 ℃, and were capable of producing oxidase and pyocyanin. The plate antagonism assay results showed that the inhibition rates of SX6 and ZJ10 against R. solanacearum were 124.87% and 63.08%, respectively. The 28-day pot experiments showed that the protective rates of SX6 and ZJ10 were 42.85% and 28.57%, respectively. LC-MS results showed that pyocyanin was one of the main secondary metabolites of SX6 and ZJ10. The inhibition rates of the crude pyocyanin extracts of SX6 and ZJ10 against R. solanacearum were 28.76% and 25.45%, respectively.Conclusion The pathogen causing bacterial wilt in Rucheng yellow ginger is R. solanacearum. The biocontrol P. aeruginosa strains SX6 and ZJ10 are isolated from ginger stem exudates, and pyocyanin could be the main antagonistic substance.