生姜青枯病生防菌株 NX 7 的鉴定、抗菌活性物质分析及发酵条件优化

Identification, optimization of fermentation conditions and analysis of antibacterial substances of biocontrol strain NX 7 against Ralstonia solanacearum

摘要

青枯病是生姜栽培产业最为严重的土传病害之一，传统化学防治存在防效差、环境污染等诸多弊端。为制定生姜青枯病生防措施，本研究于生姜根际分离获得 1 株对青枯病菌拮抗效果较好的菌株 NX 7，经形态学观察、生理生化特性测定并结合分子生物学鉴定明确其分类地位，通过液质联用技术（LC-MS）对其抑菌产物进行初步分析，并采用琼脂扩散法，结合单因素试验及正交试验确定其最适发酵培养基组分、发酵条件，分析优化后菌株抑菌效果。结果表明，菌株 NX 7 鉴定为贝莱斯芽孢杆菌（ Bacillus velezensis），抑菌圈直径为 1.15 cm，其发酵液内含有 Minocycline、Lankacidin C 及 Rifaximin 等 3 种聚酮类代谢产物，其中 Rifaximin 抑菌活性最强，EC₅₀为 287.66 μg·mL^-1。单因素试验及正交试验结果显示，菌株 NX 7 最适培养基组分为蛋白胨 11 g·L^-1、牛肉浸粉 7 g·L^-1及硫酸锌 11 g·L^-1，最适发酵条件为培养基初始 pH 6、培养温度为 25 ℃、转速为 180 r·min^-1。优化后菌株 NX 7 抑菌圈直径较常规处理提高 93.48%。综上所述，菌株 NX 7 对生姜青枯病病菌具有较强的抑菌效果，经发酵培养基及发酵条件优化后可增强其抑菌能力。菌株 NX 7 具有开发成生姜青枯病生防菌剂的潜力。

Abstract

Bacterial wilt of ginger caused by Ralstonia solanacearum, is the destructive pathogen of ginger throughout the entire growth cycle. Traditional chemical control methods have many drawbacks such as poor efficacy and environmental pollution. Thus, it is urgent to establish an environmentally friendly and highly efficient biological control system. To investigate the resistance potency of plant growth-promoting rhizobacteria in ginger to R. solanacearum, one strain, marked as NX 7, was isolated from ginger rhizosphere soil and exhibited potent antagonistic effects against R. solanacearum. Morphological observations, physiological and biochemical examination, and molecular biological identification were deployed to classify the antagonist strain. The antibacterial activity of NX 7 strains was quantified through agar diffusion method. Single-factor experiments and orthogonal tests were subsequently conducted to optimize the fermentation medium components and fermentation conditions. The strain NX 7 was identified as Bacillus velezensis based on the multiple methodology. It formed an inhibition zone of 1.15 cm in diameter and produced Minocycline, Lankacidin C and Rifaximin in fermentation broth. Meanwhile, the Rifaximin exhibited the strongest inhibition effect on the pathogen and EC ₅₀ is 287.66 μg·mL ^-1. The results of single factor and orthogonal experiments showed optimal growth medium for strain NX 7 was contained peptone（11 g·L^-1）, beef extract desiccant（7 g·L ^-1）, and zinc sulfate（11 g·L ^-1）. The best fermentation conditions were determined as initial pH of 6.0, temperature of 25 ℃, rotation speed of 180 r·min ^-1. The diameter of inhibition zone of the optimized strain NX7 against R. solanacearum increased to 3.56 cm, which was 93.48% larger than fermentation medium conditions. In summary, the strain NX 7 exhibited a strong antagonist activity against R. solanacearum. Optimized medium and fermentation conditions enhanced the antibacterial activity of NX 7 strain, which demonstrated its potential as a biocontrol agent.

关键词

生姜 / 青枯病 / 贝莱斯芽孢杆菌 / 发酵条件 / 正交试验 / 聚酮类代谢产物

Key words

Ginger / Bacterial wilt / Bacillus velezensis / Fermentation condition / Orthogonal test / Polyketide metabolite

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基金资助

山东省自然科学基金(ZR2023QC326)