Nano silver-chitosan resonates with the zineb fungicide that causes dragon fruit white spot disease
Nano Silver – chitosan (Ag @ CS) as a new drug delivery system have been developed for fungicides. In this study, the synergistic effects of silver nanoparticles (AgNPs), chitosan (CS), and the fungicide zineb (Zi) were studied as antifungal material against Neoscytalidium dimidiatum in dragon fruit. More specifically, Ag @ CS is prepared by dipping AgNPs into CS polymers and then combined with Zi. Transmission electron microscopy was used to confirm the morphology and size of Ag @ CS. The diameter of the spherical nanoparticles is about 4.11 ± 0.37 nm. Furthermore, the nano silver chitosan formation was characterized by Fourier transform X-ray diffraction and infrared analysis. The thermal stability properties of these nanoparticles were also determined by thermal gravimetric analysis. In particular, the antifungal activity of Ag @ CS has been shown to be better antifungal than the individual components, analyzed according to the inhibitory zone method for N. dimidiatum. These results show potential applications of Ag @ CS in the development of nanomaterials for the treatment of agricultural diseases.
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1. Introduce
Dragon fruit or pitahaya (Hylocereus undatus) is commonly known as a nutritious fruit, becoming the main crop with large area, high income. It is considered a good source of potassium, phosphorus, vitamin C (ascorbic acid), calcium, carbohydrates and fiber, which may reduce the risk of stroke and coronary heart disease by preventing high cholesterol and improving the system. Digested with its fiber and also avoids cancer. Up to the present time, Vietnam is one of the countries supplying dragon fruit on a commercial scale to China, Hong Kong, Japan, Singapore and many countries in Europe.
According to the Department of Crop Production, Ministry of Agriculture and Rural Development, dragon fruit growing area is more than 28,700 hectares with an output of 520,000 tons / year, accounting for 61.4% of total fresh fruit exports [1], [ 2 ]. However, when growing dragon fruits on a large area, farmers face many difficulties in preventing diseases caused by microorganisms. Neoscytalidium dimidiatum, the pathogen of pitahaya brown spot, is one of the dangerous plant pathogens, causing great damage to agricultural crops, especially on dragon fruit.
It not only affects the fruit yield for export but also the economic result. To prevent brown rot, chemical drugs are commonly used in agriculture due to their therapeutic effects [3], [4], [5]. However, incorrect use of the drug or ineffective dosage can prolong treatment time and reduce crop yield. In addition, low quality chemicals can lead to harmful consequences, increasing production costs, low control, low productivity, and low profit [6], [7]. Moreover, disease outbreaks mean that farmers often use high doses of antibiotics, leading to an excess of the drug level, which can destroy beneficial insects in the soil and create resistance to pathogens [8 ], [9], [10], [11]. Consequently, the yield and quality of crops are reduced, negatively affecting the environment and human health [6], [12], [13].
Recently, silver nanoparticles (AgNP) have been combined with various nanofibers and drugs to increase antimicrobial efficacy for antimicrobial applications. Since the early twentieth century, Ag has been used as one of the typical ingredients in alternative medicine and functional foods [14], [15], [16], [17], [18]. It has also been used in the photographic industry since the 1800s, disinfecting drinking water and disinfecting swimming pools [19], [20].
Furthermore, Kokura [21] reported that low concentrations of AgNPs have sufficient preservative efficacy against bacteria and fungi and do not penetrate normal human skin in cosmetic application. Importantly, the antibacterial activities of AgNPs were also studied successfully in Kyung-Hwan Cho’s study at 5–10 ppm [22] and at 20 ppm in Sotiriou’s study [23].
Several independent cell tests have been completed on AgNPs products from American Biotech Laboratories. At 10–22 ppm, no damage was found when the AgNPs products were tested with human or monkey cells, meaning they were completely cytotoxic [24], [25], [26] ].
In addition, chitosan (CS), an abundant natural hydrophilic biotic agent, is often selected for combination with AgNPs due to its unlimited biological compatibility, biodegradability, and better stability. and low toxicity [8], [27], [28], [29], [30]. Many studies have shown that this polymer acts as plant growth stimulant that helps plants fight infection from microbial activity [8], [31], [32], [33], and is resistant to infection by microorganisms. good bacteria [8], [34], [35], [36], and stimulates the immunity of the plant system [37], [38]. With these advantages, the combination of CS and AgNPs (Ag @ CS) is a potential approach for antimicrobial goals.
As a result, bio-nano complexes exhibited higher antimicrobial activity than any single active ingredient. This system is notable for being one of the extended applications of nano silver chitosan that can act as a safe fungicide for nanotechnology and agriculture.
In this study, zineb (Zi) combined with nano silver chitosan (Ag @ CS-Zi) against N. dimidiatum was developed. The synthesis of AgNP encased in biopolymers CS has been studied for the production of bio-nano compounds with enhanced antifungal properties. Research focused on the synthesis of nano silver chitosan and testing their fungicidal properties. In addition, the nanoparticles were characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and thermal gravity analysis (TGA).
Furthermore, the antifungal activities of the composite against fungi were examined using the inhibitory zone method on potato dextrose agar plates (PDA). The expected result is that the amount of CS, Ag and Zi used will decrease compared to the normal amount while the antifungal ability remains as expected. This study is expected to produce a significant antimicrobial drug, a fungicide that is safe for agriculture.
2. Materials and methods
2.1 Materials
Chitosan was purchased from Sigma Aldrich Co. (St. Louis, MO, USA) (CS, USD 150,000 million, 75–85% demineralized), acetic acid (CH 3 COOH), silver nitrate (AgNO 3) and sodium hydroxide (NaOH) purchased from Sigma -Aldrich (St. Louis, MO, USA). Zi Bul 80 WP was purchased from Saigon Plant Protection Joint Stock Company (Ho Chi Minh City, Vietnam) (80% Zi C 4 H 6 N 2 S 4 Zn) purchased from Security Joint Stock Company Saigon Plants (Ho Chi Minh City, Vietnam). All reagents and solvents were used without further refinement.
2.2 Methods
2.2.1 Synthesize Ag @ CS and Ag @ CS-Zi:
Ag @ CS is synthesized using CS as a reducing and protection polymer and AgNO 3 with NaOH by chemical reduction. Initially, CS (0.2 g, dissolved in 10 ml of 1% volume CH3 COOH solution, pH 3.5) was mixed with AgNO 3 (2.5 ml, 1.0 × 10 −2 m). under continuous stirring for 30–45 minutes. After this step, a mixture of AgNO 3 and CS solution is obtained. Next, NaOH solution (10 ml, 1 m) is dropped into the mixture by a syringe pump. After 15–20 minutes, the golden brown spheres were rinsed to remove the residue, and Ag @ CS was placed in the refrigerator to prevent Ag reduction from continuing. Finally, Zi was combined with Ag @ CS by soaking it in water at room temperature under continuous stirring for 24 hours. A volume ratio of 2: 1 between Zi and Ag @ CS solution was used in the synergy process.
2.2.2 Antifungal effects of Neoscytalidium dimidiatum:
To test the antifungal effect, the paper plate method was used in this study. First, a PDA agar plate (infusion of potatoes at 200.00 g / l, dextrose 20.00 g / l, and agar 15.00 g / l) was prepared. Second, the cell suspension containing fungal colonies is spread directly on the agar surface. Then, circular filter paper (diameter 5 mm) containing AgNP, CS and the dissolved Ag @ CS compound was inserted into the plate. The inhibitory area diameter is measured. The difference between the control and the treated samples was checked for significance using the test (p <0.05).
2.2.3 Characteristics:
Morphological characteristics of the TEM-synthesized nanoparticles were determined using FEI Tecnai G2 20 S-Twin at 100 kV. To characterize the thermal decomposition profiles of CS or Ag @ CS composites, TGA was performed on TGA (Q500 V20.10 Bluid 36) and 5 mg samples of both composites were measured at nitrogen flow degrees at heating rates of 10 ° C / min -1 from 30 ° C to 800 ° C. FT-IR spectra were recorded using the FT-IR Spectrum Tensor27, using the FT-IR Spectrophotometer KBr tablets in the range 500–4500 cm −1 with a resolution of 4 cm −1. XRD samples were obtained at room temperature using Cu K-α radiation (λ = 1.5406 Å) with a range of 2θ = 10 ° –90 ° and scanning rate 0.03 s −1. AgNPs were prepared by adding 20 μl CH 3 COOH solution and 1 ml H 2 O distilled to Ag @ CS and then swirled for 3-5 minutes. Ag @ CS was dropped into the net with a micropipette for further experiments.
3 Results and Discussion
3.1 Characteristics of nanoparticles
3.1.1 Seed size and morphology
Morphology and dimensions of nano silver chitosan were provided by TEM (Fig. 1). The well-separated and properly dispersed particles are spherical and not more than 5 nm in size. The size of Ag @ CS was measured to obtain an appropriate mean diameter of 4.11 ± 0.37 nm, and this is also consistent with XRD samples. The size distribution of the particles indicates high dispersion. The synthesized nanoparticles completely separate from each other without clumping, increasing the effectiveness of their interactions with drugs and the surrounding environment. Compared to the shape of AgNP and CS, Ag @ CS has no significant difference in shape and size distribution, which means CS can act as a good stabilizer to prevent AgNP agglutination. and cause them to have a narrow size distribution during cross-linking [32], [39]. During the synthesis of AgNPs, without any other preservative chemicals, CS acts as a dispersing agent that removes the agglomeration of the particles. In 2011, M. Carmen Rodriguez surveyed the average AgNPs below 2 nm [40]. This size is quite small for refills or in combination with other ingredients. The physical and chemical properties of the nanoparticles depend on their size and surface properties. According to previous reports, the nanoparticles range in size from 4 nm to 10 nm, allowing them to interact with their surroundings because of their permeability and retention enhancing effects. The average particle size of the almost spherical nanoparticles is 4 nm, which is the right size for the particles to synergize and ensure stability and specificity for their end purpose. Thanks to that, the efficiency is significantly increased.
Figure 1 TEM image (A) and particle size distribution (B) of Ag @ CS.
3.1.2 FT-IR analysis
The existence of chemical bonds between CS and AgNPs was examined by FT-IR (Figure 2). The characteristic peaks of Ag @ CS at 3421 cm −1 and CS at 3424 cm −1 were consistent with prolonged fluctuations of NH-amines in the amino groups. The CH alkanes elongating lipids, namely -CH 2 and -CH 3 groups CS and Ag @ CS, are shown in the ranges 2920 cm −1 to 2924 cm −1. Strips of CS at 1658 cm −1 and that of Ag @ CS at 1630 cm −1 are related to amide groups of amides. Peak C = C of aromatic amine groups linked to a peak of 1380 cm −1. Strips between 1076 cm −1 and 1085 cm −1 are indicated as carbonyl elongated proteins present in CS. It can be seen that through the free amino groups, protein binding AgNPs are stabilizers that reduce AgNO 3 to AgNPs during treatment. In addition, the AgNPs peak at 1492 cm −1 and -NH 2 elongated at 1423 cm −1 of CS shifted to 1380 cm −1 in the nanoparticle synthesis curve. It illustrates that the -NH 2 or -OH groups of CS are combined with Ag + / Ag 0 via electrostatic bonding. This result has also been confirmed through the study of Vigneshwaran [41]. Furthermore, the band of the OH group at 3421 cm −1 and the elongation of the main amine at 1659 cm -1 were not shown, indicating that Ag binding to the nitrogen atom. These results show the presence of an electrostatic bond between CS and Ag, creating an Ag @ CS mixture.
Figure 2 FT-IR spectra of nano silver chitosan (A) and CS (B).
3.1.3 Analysis of X-ray diffraction
Figure 3 illustrates the Ag @ CS XRD patterns. The wide reflection at 9 ° is due to the crystallization of CS in the composites. In addition, the Bragg reflections at 38.16 °, 46.12 °, 64.09 °, 77.01 °, and 86 ° are assigned to diffractions from indexed planes (2,336), ( 1,947), (1,380), (1,237) and (1,129) of metallic Ag nanoparticles. The AgNPs form and the pure CS result showed the unchanged crystal structure of AgNPs after being applied to CS polymers.
Figure 3. Ag @ CS XRD pattern.
3.1.4 Temperature measurement analysis
To analyze the thermal stability of nano silver chitosan and CS, TGA is shown in figure 4. In the range of 50–800 ° C, the samples show two distinct stages of weight loss: (1) From 50 to 150 ° C, 10% of weight was lost due to water evaporation in the Ag @ CS sample (50–100 ° C), while CS, which was dried, remained weight loss at temperatures higher than 100 ° C (low than normal decomposition of CS). This is because the initial CS not only escapes from the long polymer chain but also many short CS oligomer leads to short thermal stability; (2) from 150 to 800 ° C, the thermal decomposition of the Ag @ CS complex was lower than that of CS. These results indicate that when dipping AgNPs into CS polymers, Ag can break the structure of CS during dissolution in acids and bases; therefore, the weight reduction of CS acting on Ag was higher than the initial CS.
Figure 4. Thermometric curves of CS (A) and Ag @ CS (B).
3.2 Antifungal activity test
The antifungal activity of Ag, Zi, Ag @ CS and Ag @ CS-Zi was estimated for N. dimidiatum by agar diffusion method. The white control plate is a pure cell suspension without any other ingredients. To compare with the effective antifungal effect, four different concentrations of Ag, Zi, Ag @ CS and Ag @ CS-Zi were used as the comparison sample (Table 1).
Table 1. Diameter of inhibited region of Ag, Ag @ CS and Ag @ CS-Zi.
According to the table, antifungal resistance is quite low when using AgNPs only. However, when Ag was combined with CS, the fungicidal capacity increased significantly (Figure 5). The diameter of the inhibitory zone is 15.00 ± 0.00 mm at 5 ppm, completely better than the diameter with concentration at 10 ppm (13.33 ± 0.58 mm). When Zi was combined with Ag @ CS, antifungal resistance was significantly increased. While pure Zi at 500 ppm and 1000 ppm was not able to remove fungi, the inhibitory zone of Ag @ CS-Zi increased to 12.00 ± 0.00 mm, almost the same diameter when using 5 ppm Ag ( 12.33 ± 0.58 mm) and more when using 5000 ppm of Zi (9.00 ± 0.00 mm). The inhibitory zone diameter of Ag @ CS-Zi at 2500 ppm of Zi was 20.67 ± 0.58, indicating its high antifungal activity compared to the individual components. In particular, the synergistic effects of Ag @ CS at 2 ppm and Zi at 5000 ppm show strong and high inotropic resistance. After a series of experiments,
Figure 5. Mean observed inhibitory regions of AgNPs (A), Zi (B), Ag @ CS (C) and Ag @ CS-Zi (D) against Neoscytalidium dimidiatum.
4. Conclusion
In this study, AgNPs were combined with functionalized CS, helping to improve stability and synergize with the fungicide to enhance antifungal activity. According to the analysis results of FT-IR and XRD, nano silver chitosan containing fungicide has been successfully synthesized. TEM images show that the Ag @ CS is almost spherical with a diameter of 4.11 ± 0.37 nm. Research has proven that Ag @ CS has good results in killing fungi. Ag’s anti-fungal ability increases the fungicidal effect of CS. Based on nano silver chitosan, new approaches for a variety of antifungal, antibacterial, antiviral and other substances can be developed and improved. This is considered an important step of antibiotics in agriculture to contribute to reducing the amount of pesticides and eliminating harmful products.
Reference source: Synergistic antifungal effect of fungicide and chitosan-silver nanoparticles on Neoscytalidium dimidiatum