Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (14): 2885-2896.doi: 10.3864/j.issn.0578-1752.2020.14.012

• PLANT PROTECTION • Previous Articles     Next Articles

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

XIANG ShunYu1,3(),WANG Jing1,XIE ZhongYu1,SHI Huan1,CAO Zhe1,JIANG Long1,MA XiaoZhou1,3,WANG DaiBin4,ZHANG Shuai5,HUANG Jin2,3(),SUN XianChao1,3()   

  1. 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 E-mail:xiangshunyu0325@163.com;huangjin2015@swu.edu.cn;sunxianchao@163.com

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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

Fig. 1

Uv-vis (A) and XPS (B) spectrum of S-AgNPs"

Fig. 2

Morphology and stability characterization of S-AgNPs A、B：TEM image of S-AgNPs solution;C、D：TEM image of S-AgNPs solution re-dissolved in water after freeze-drying;E、F：SEM images of S-AgNPs;G、H：AFM images of S-AgNPs;I：TEM particle size statistics of S-AgNPs;J：AFM particle size statistics of S-AgNPs"

Fig. 3

Zeta particle size statistical graph (A) and Zeta potential graph (B) of S-AgNPs"

Fig. 4

Antifungal effect of S-AgNPs on A. alternata (A) and inhibition rate (B) Different lowercases on the bars indicate significantly different among different treatments according to the Duncan’s multiple range test (p<0.05). Vertical bars indicate standard deviations. The same as below"

Fig. 5

Effect of S-AgNPS on mycelia growth A、B：Healthy mycelium and S-AgNPs treated mycelium;C：Dry weight of mycelium;D：Fresh weight of mycelium"

Fig. 6

SEM images of mycelia of the healthy group and S-AgNPs treatment group"

Fig. 7

Mycelium conductivity change (A) and DNA leakage (B)"

Fig. 8

Soluble protein content of mycelium"

Fig. 9

Incidence of tobacco brown spot under different treatments"

Table 1

Disease index of tobacco brown spot under different treatments"

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

Table 2

Toxicity of S-AgNPs to crucian carp"

处理
Treatment		 死亡数量 Number of deaths
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
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