一种新型银纳米颗粒的制备及其抑制烟草赤星病菌的机制

doi:10.3864/j.issn.0578-1752.2020.14.012

中国农业科学 ›› 2020, Vol. 53 ›› Issue (14): 2885-2896.doi: 10.3864/j.issn.0578-1752.2020.14.012

一种新型银纳米颗粒的制备及其抑制烟草赤星病菌的机制

向顺雨^1,³(),王靖¹,谢中玉¹,施焕¹,曹哲¹,江龙¹,马小舟^1,³,汪代斌⁴,张帅⁵,黄进^2,³(),孙现超^1,³()

¹西南大学植物保护学院,重庆 400715
²西南大学化学化工学院,重庆400715
³西南大学软物质材料化学与功能制造重庆市重点实验室,重庆400715
⁴重庆烟草科学研究所,重庆400715
⁵重庆市烟草公司酉阳分公司,重庆409800

收稿日期:2020-01-02 接受日期:2020-02-15 出版日期:2020-07-16 发布日期:2020-08-10
通讯作者: 黄进,孙现超
作者简介:向顺雨,E-mail：xiangshunyu0325@163.com。
基金资助:
国家自然科学基金(31670148);国家自然科学基金(31870147);中央高校基本科研业务费专项资金(XDJK2020B064);中国烟草总公司重庆公司科技项目(NY20180401070010);中国烟草总公司重庆公司科技项目(NY20180401070001);中国烟草总公司重庆公司科技项目(NY20180401070008)

Preparation of A Novel Silver Nanoparticle and Its Antifungal Mechanism Against Alternaria alternata

XIANG ShunYu^1,³(),WANG Jing¹,XIE ZhongYu¹,SHI Huan¹,CAO Zhe¹,JIANG Long¹,MA XiaoZhou^1,³,WANG DaiBin⁴,ZHANG Shuai⁵,HUANG Jin^2,³(),SUN XianChao^1,³()

¹ College of Plant Protection, Southwest University, Chongqing 400715
² School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715
³ Chongqing Key Laboratory of Soft-Matter Material Chemistry and Function Manufacturing, Southwest University, Chongqing 400715
⁴ Chongqing Tobacco Science Research Institute, Chongqing 400715
⁵ Chongqing Tobacco Company Youyang Branch, Chongqing 409800

Received:2020-01-02 Accepted:2020-02-15 Online:2020-07-16 Published:2020-08-10
Contact: Jin HUANG,XianChao SUN

摘要/Abstract

摘要：

【目的】利用生物多糖合成银纳米颗粒,并解析其对烟草赤星病菌（Alternaria alternata）的抑制活性及机理,探索生物多糖合成银纳米颗粒的农业应用前景,为开发安全、高效的抗菌剂提供理论依据。【方法】采用海藻酸钠作为还原剂与表面活性剂在水浴锅中（65℃）一步合成银纳米颗粒（S-AgNPs）。利用透射电镜（TEM）、扫描电镜（SEM）、原子力显微镜（AFM）、紫外分光光度计（UV-vis）、Zeta电位-粒径仪和X射线光电子能谱分析（XPS）分别对S-AgNPs粒径、分散性、稳定性及化学组成进行表征分析。应用Nano Measure软件统计TEM与AFM图像中S-AgNPs的平均粒径。采用平板生长速率法探究S-AgNPs在PDA培养基中浓度为0.0625、0.125、0.25、0.5、1.0 μg·mL^-1时对赤星病菌菌丝生长的影响。赤星病菌在S-AgNPs浓度为1.0 μg·mL^-1的液态PDA培养基（PDB）中培养后,分别通过测定菌丝的鲜重与干重、SEM观察、电导率测定和考马斯亮蓝G250染色,分析S-AgNPs对菌丝生长量、形态、细胞膜通透性和菌丝可溶性蛋白含量的影响。用孢子悬浮液注射接种法测试S-AgNPs在离体烟草叶片上对赤星病的防治效果。最后通过测定S-AgNPs对鲫鱼生长的影响分析海藻酸钠合成的银纳米颗粒的安全性。【结果】S-AgNPs的TEM、SEM、AFM图像分析表明该方法所合成的S-AgNPs具有分散性好,粒径统一,稳定性强等特点,平均粒径为9.83 nm。生测结果显示S-AgNPs在1.0 μg·mL^-1时对烟草赤星病菌菌丝生长抑制率为83.9%。S-AgNPs处理组的菌丝鲜重与菌丝干重明显低于清水对照组,S-AgNPs可以明显破坏菌丝表面结构。进一步抑菌机制研究表明S-AgNPs主要通过抑制赤星病菌可溶性总蛋白合成而破坏其生物膜结构,降低生物膜保水能力,快速破坏菌丝细胞膜通透性,造成大量细胞质渗漏而抑制菌丝生长发育。最终,离体试验证实S-AgNPs能够有效地抑制烟草赤星病菌侵染烟草叶片,且S-AgNPs在有效浓度（1.0 μg·mL^-1）内对鲫鱼无明显毒害作用,不影响其正常生命活动。【结论】建立的方法能够稳定合成分散性好,粒径统一,稳定性强的银纳米颗粒。该银纳米颗粒对烟草赤星病菌具有较强的抑制作用,可有效防治烟草赤星病,具有潜在的田间应用前景。

关键词: 银纳米颗粒, 海藻酸钠, 绿色合成, 烟草赤星病菌, 烟草赤星病, 抗菌机制

Abstract:

【Objective】In order to explore the agricultural application prospect of biological polysaccharide synthesis of silver nanoparticles and provide a theoretical basis for the development of safe and efficient antifungal agent, the silver nanoparticle based on the polysaccharide was prepared and its inhibitory activity and mechanism against Alternaria alternata was analyzed.【Method】The sodium alginate was used as reductant and surfactant to synthesize the silver nanoparticles in water bath (65℃). The transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), ultraviolet spectrophotometer (Uv-vis), Zeta potential-particle size analyzer and X-ray diffraction energy spectrum (XPS) were used to characterize and analyze the particle size, dispersion, stability and chemical composition of S-AgNPs, respectively. The Nano Measure software was used to measure the mean particle size of S-AgNPS in TEM and AFM images. Then, the S-AgNPs inhibition ability against mycelial growth was tested in the potato dextrose agar medium (PDA) with different S-AgNPs concentrations (0.0625, 0.125, 0.25, 0.5, 1.0 μg·mL^-1). After A. alternata cultivated in the liquid PDA medium (PDB) with S-AgNPs at the concentration of 1.0 μg·mL^-1, the effects of S-AgNPs on the mycelium weight, morphology, the cell membrane permeability and the soluble protein content were investigated by testing the fresh and dry weights of mycelia, SEM observation, conductivity measurement and coomassie brilliant blue G-250 solution stain. Then, the control efficacy of S-AgNPs on tobacco brown spot was tested in vitro leaves by the spore suspension injection method. Finally, crucian carp was used to evaluate the biosafety of S-AgNPs.【Result】The TEM, AFM and SEM images showed that the strategy could be used to well control the size of the S-AgNPs (average particle size of 9.83 nm), and the prepared S-AgNPs showed a high stability and dispersibility in aqueous solvent. The results of bioassay showed that the inhibition rate of S-AgNPs on the growth of A. alternata reached 83.9% at 1.0 μg·mL^-1. The fresh weight and dry weight of mycelium in S-AgNPs treatment group were significantly lower than those of the control group. The SEM image of mycelium showed that S-AgNPs could obviously destroy mycelium surface structure. Further investigation on the antifungal mechanism of S-AgNPs showed that S-AgNPs could destroy the biofilm structure by inhibiting the synthesis of soluble total protein in A. alternata, reducing the water retention capacity of biofilm, rapidly destroying the membrane permeability of mycelium and causing a large amount of cytoplasmic leakage to inhibit the growth and development of mycelium. Finally, in vitro experiments confirmed that S-AgNPs could effectively inhibit A. alternata infecting tobacco leaves. Moreover, S-AgNPs in the effective antifungal concentration had no toxic effect on crucian carp and did not affect its normal life.【Conclusion】The method established in this study can stably synthesize silver nanoparticles with good dispersion, uniform particle size and strong stability. In addition, the silver nanoparticle has a strong inhibitory ability against A. alternata and can effectively control tobacco brown spot, which suggests that S-AgNPs has a potential application prospect.

Key words: silver nanoparticle, sodium alginate, green synthesis, Alternaria alternata, tobacco brown spot, antifungal mechanism

向顺雨,王靖,谢中玉,施焕,曹哲,江龙,马小舟,汪代斌,张帅,黄进,孙现超. 一种新型银纳米颗粒的制备及其抑制烟草赤星病菌的机制[J]. 中国农业科学, 2020, 53(14): 2885-2896.

XIANG ShunYu,WANG Jing,XIE ZhongYu,SHI Huan,CAO Zhe,JIANG Long,MA XiaoZhou,WANG DaiBin,ZHANG Shuai,HUANG Jin,SUN XianChao. Preparation of A Novel Silver Nanoparticle and Its Antifungal Mechanism Against Alternaria alternata[J]. Scientia Agricultura Sinica, 2020, 53(14): 2885-2896.

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处理Treatment	病情指数Disease index
CK	100a
0.125 μg·mL^-1	48.1b
0.25 μg·mL^-1	11.1c
0.5 μg·mL^-1	0d
1.0 μg·mL^-1	0d

处理 Treatment	死亡数量 Number of deaths
处理 Treatment	24 h	48 h	72 h	96 h
水 Water	0	0	0	1
S-AgNPs (1.0 μg·mL^-1)	0	0	1	1

一种新型银纳米颗粒的制备及其抑制烟草赤星病菌的机制

Preparation of A Novel Silver Nanoparticle and Its Antifungal Mechanism Against Alternaria alternata

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