耐高盐枯草芽孢杆菌XP合成球形纳米硒及其抑制草莓病原真菌生物活性

Biosynthesis of spherical selenium nanoparticles with halophilic Bacillus subtilis subspecies stercoris strain XP for inhibition of strawberry pathogens

摘要:

生物方法合成纳米材料具有低能耗、高安全性以及环境友好等优良特点，因而备受人们关注。利用细菌将硒酸盐或亚硒酸盐还原为单质硒，不仅可以降低硒毒性，而且还能获得价值更高的生物纳米材料。文中选用可耐受高盐环境胁迫的枯草芽孢杆菌亚种Bacillus subtilis subspecies stercoris strain XP构建生物模型，分别以LB液体培养基和亚硒酸钠为介质和底物 (电子受体)，解析菌株XP合成纳米硒的基本规律。通过扫描电镜 (Scanning electron microscope，SEM) 观察、X射线能谱分析 (X-ray energy dispersive spectral analysis，EDAX)、X射线衍射 (X-ray diffraction，XRD) 分析、傅里叶红外变换光谱 (Fourier transform infrared spectroscopy，FTIR) 技术对合成的纳米硒进行物理化学表征分析，同时选用草莓枯萎、红叶、紫斑病病原真菌对其抗菌活性进行分析。结果表明，菌株XP介导合成的单质硒为球形纳米颗粒 (Selenium nanoparticles，SeNPs)，其生成量与反应时间呈正相关 (0–48 h)，且细胞形态未发生褶皱或破损等变化 (耐受力强)；SeNPs为非晶态，粒径范围在135–165 nm，表面元素组成以Se为主，同时存在C、O、N、S等有机元素；颗粒表面包裹生物大分子物质，-OH、C=O、N-H、C-H等官能团与SeNPs稳定性和生物活性密切相关；高浓度纳米硒对枯萎、红叶、紫斑病病原真菌均有显著抑制活性 (P<0.05)，其中对草莓红叶病与枯萎病病原真菌的抑制活性明显优于对紫斑病病原真菌的抑制活性。总而言之，菌株XP不仅耐受高盐胁迫能力强，同时还可介导合成生物SeNPs，其合成的纳米硒颗粒具有良好的稳定性和生物活性，在草莓病害防治以及绿色富硒草莓种植等领域具有潜在的应用价值。

关键词:枯草芽孢杆菌，生物纳米硒，物理化学表征，抑菌活性

Abstract:

Biosynthesis of nanomaterials has attracted much attention for its excellent characteristics such as low energy consumption, high safety, and environmental friendliness. As we all know, the toxic selenite can be transformed into higher-value nanomaterials by using bacteria. In this study, nano-selenium was synthesized by halophilic Bacillus subtilis subspecies stercoris strain XP in LB medium supplemented with selenite (electron acceptor). The physicochemical characteristics of nano-selenium were analyzed by scanning electron microscope (SEM), X-ray energy dispersive spectral analysis (EDAX), X-ray diffraction (XRD), and fourier transform infrared spectroscopy (FTIR). Meanwhile, the antifungal activity of nano-selenium to strawberry pathogens (fusarium wilt, erythema, and purple spot fungi) was determined. The products from reduction of selenite by strain XP was amorphous spherical selenium nanoparticles (SeNPs) with a diameter range of 135–165 nm. The production of SeNPs was positively correlated with time (0–48 h) and no changes were observed on cell morphology. Selenium was dominant in the surface of SeNPs where the organic elements (C, O, N, and S) existed at the same time. SeNPs were coated with biomolecules containing functional groups (such as -OH, C=O, N-H, and C-H) which were associated with the stability and bioactivity of particles. Although the highest concentration of SeNPs had significant (P<0.05) inhibitory effects on three strains of strawberry pathogens, antifungal activity to erythema and fusarium wilt pathogenic fungi was higher than that to purple spot pathogenic fungi from strawberry. In conclusion, strain XP not only has strong tolerance to high salt stress, but can be also used to synthesize biological SeNPs with good stability and biological activity. Thus, the strain XP has bright perspectives and great potential advantage in pathogens control and green selenium-rich strawberry planting as well as other fields.

Key words:Bacillus subtilis subsp., biological nano-selenium, physicochemical characteristics, antifungal activity