Nano silver (AgNPs) have been successfully prepared by irradiating solutions containing 1.0–10 mM silver nitrate and 1% (10000 ppm) chitosan. The optical properties and particle size of AgNPs were determined by UV-Vis spectroscopy and TEM images, respectively. The size of AgNPs increases with increasing silver concentration or decreasing the molecular weight of chitosan in the irradiated solution. The in vitro test crystals showed that AgNPs inhibited the growth of Corynespora cassiicola on rubber leaf extract with an inhibitory effect of 52.1-100% by treating AgNPs with a particle size of 15-5 nm. , corresponding. In addition, antifungal activity reached ~ 100% with the addition of 90 ppm AgNPs. In vivo, foliar treatment of AgNPs on 9-month-old rubber trees showed that treatment with 2.5–12.5 ppm AgNPs in experimental plants after transplanting by spraying C. cassiicola spores improved the rate. The rate of uninfected plants ranged from 6.0 to 93.3%, respectively, compared with the untreated control. The inhibitory effect of AgNPs on C. cassiicola growth was also elucidated by SEM imaging. Irradiated AgNPs / chitosan has the potential to be used as a fungicide for the treatment of C. cassiicola, a fungus that causes serious diseases on rubber trees.
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Rubber tree (Hevea brasiliensis) is a perennial industrial tree and provides raw materials for many industries. They have a very high economic value and bring a lot of profits to agriculture. According to the General Statistics Office of Vietnam in 2017, Vietnam ranked third with a rubber area of 971.6 thousand hectares and a total rubber latex production of 1,086 thousand tons. However, at present, rubber growers are facing many difficulties due to many diseases caused by microorganisms, in which defoliation caused by C. cassiicola is seriously affecting the growth and yield of rubber. [first].
Cassiicola disease was first observed on rubber trees in Sierra Leone (Affrica) in 1936. More cases have been reported than in. India and Malaysia in 1961; Nigeria in 1968; Thailand, Sri Lanka and Indonesia in 1985; Brazil and Bangladesh in 1988 [2, 3]. Although rubber defoliation has only appeared in Vietnam since August 1999, the disease has spread rapidly and widely in many provinces in the Southeast, Central Highlands and Central Coast of Vietnam.
Deficiency control is still a difficult problem due to the lack of specific fungicides while growers are using many chemical products that can have many negative environmental and quality impacts. rubber.
The low molecular weight chitosan has been shown to be a natural, safe and effective product for agriculture . Many studies have been reported that in addition to its growth enhancing effects, chitosan also gives plants the ability to prevent many infections by strengthening the immune system of plant cells, known as is phytoalexin effect [5 – 7].
In addition, silver nanoparticles (AgNPs) have been widely studied and used extensively for a long time due to their own properties such as inhibiting bacteria and fungi, deodorizing at low concentrations, and safety for children. people and the environment. The antifungal effect of nano silver stabilized in chitosan has been studied in pathogenic fungi such as Colletotrichum  and Corticium salmonicolor .
Furthermore, γ -rays irradiation using a Co-60 source is considered to be an effective method for the synthesis of precious metal nanoparticles . The advantage of the irradiation method is that it saves energy, space and materials, is environmentally friendly, etc. In particular, the process can be carried out at ambient temperature and is favorable for mass production scaling at reasonable cost [10 – 12]. This study aimed to synthesize AgNPs / chitosan product that is safe for humans and effective in eliminating defoliation caused by C. cassiicola on rubber trees.
Materials and methods
Pure silver nitrate (AgNO3) powder supplied by Merck Co., Germany. Chitosan with 80% redox level was purchased from Loyou Chemical Co., Ltd., Japan. Pathogenic fungi, specifically Corynespora cassiicola, is a gift from the Vietnam Rubber Research Institute.
2. Synthesis of AgNPs / Chitosan by γ -Ray Irradiation
Silver nitrate solutions at 1.0 different concentrations; 2.5; 5.0; and 10 mM dissolved in 1% chitosan solution stored in glass bottles and irradiated at different doses from 8 to 28 kGy using γ -rays from Co-60 source (Gamma Chamber 5000, BRIT, India) at a dose rate of 3 kGy / NS. Silver nano colloidal solution synthesized by ray irradiation is used for further experiments.
3. Characteristics of Silver Nano / Chitosan particles
UV-Vis spectra of the obtained silver nanoparticles diluted to 0.1 mM calculated according to Ag + concentration using deionized water measured on UV-2401PC, Shimadzu, Japan [9, 11 , 13]. The size distribution and nano-silver size were determined by TEM image on a JEM 1400 gauge, JEOL, Japan, according to the method described by Li et al.  and Phu et al. .
4. Antifungal activity of AgNPs / Chitosan
Rubber leaf extract medium was used for the culture of C. cassiicola. Rubber leaf extract is prepared by boiling 200 g of fresh (disease-free) rubber leaves in 1000 ml of distilled water for 45 minutes, then filtering to remove residue and adjusting pH ~ 6.5. One liter of rubber leaf extract was mixed with 20 g of agar and added with AgNPs at different particle sizes 5; ten; and 15 nm, and the nano-silver concentration changed from 10 to 90 ppm before autoclaving at 121 ° C, 1 atm for 20 minutes. Fungal samples with a diameter of ~ 4 mm were cultured on the center of a rubber leaf extract agar plate and incubated under dark conditions at 28 ± 2 ° C. Five replicates were applied for each concentration or size text. The particle ruler and all experiments were repeated in three times. The colony diameter of C. cassiicola in the medium was measured every 24 hours and the antifungal effect of AgNPs / chitosan was calculated using the following:
Inhibitory effect (%) = (D-d)/d*100%
Where (mm) and (mm) are the diameters of the fungal colonies on the medium with or without addition of silver nanoparticles, respectively.
5. Scanning electron microscope analysis (SEM)
Approximately 5 ml of solution containing 7.5 and 12.5 ppm nano silver were sprayed on fully grown C. cassiicola mycelia grown on rubber leaf extract agar using a spray machine. Control was applied with distilled water of the same volume. All applications were carried out over a period of four days and incubated at room temperature. SEM images of the treated samples were observed on FE-SEM S4800, Hitachi Co., Tokyo, Japan, at an accelerating voltage of 10 kV.
6. In Vivo Fungicidal effect of nano silver against C. cassiicola on rubber tree
9-month-old (disease-free) rubber trees were planted and maintained in a greenhouse prior to testing. To test the elimination effect of AgNP on C. cassiicola, leaves of 210 disease-free rubber trees (30 plants per recipe) were first wound before inoculation 300 mL of C. cassiicola solution containing 10 4 fungal spores / mL. Control samples were sprayed onto leaves with the same amount of distilled water. 7 days after infection, the number of infected plants was about 50% for all treatments; 50 mL of AgNPs solution with concentrations of 2.5, 5, 7.5, 10, and 12.5 ppm were sprayed through the foliar once every 3 days, up to 3 times. The number of diseased plants and the number of diseased leaves in each treatment were observed 7 days after 28 days of the experiment. Plant and leaf disease (DI) rates are expressed as percentages (%) and are calculated using the following :
Rate of diseased plants = (Total number of diseased plants / Total number of evaluated trees) * 100%
Rate of diseased leaves = (Total number of diseased leaves / Total number of evaluated leaves) * 100%
Each test was performed three times and data were analyzed statistically using an analytical test of variance (ANOVA). The mean values were compared using Duncan multiple range test at a 5% probability level.
Results and discussion of nano silver / chitosan
1. Radiation synthesis of silver / Chitosan nanoparticles by γ-Ray irradiation
In this experiment, the solution contains silver ions of concentration 1; 2.5; 5; and 10 mM is stable in 1% chitosan with a molecular weight (Mw) of about 550 kDa irradiated at 8, 12, 16 and 28 kGy, respectively. Huang et al.  reported that different AgNP sizes lead to different maximum absorption wavelength (ƛmax) and peak intensity. The value of displacement to longer wavelengths with an increase in the size of AgNPs [9, 15, 16]. It can be seen from Figure 1 that the peak intensity values of the irradiated samples in the UV-Vis spectrum decreased from 1.26 to 1.02 due to an increase in the silver concentration in the irradiated sample from 1 to 10. mM, while the values of these samples shifted from 397 to 405.5 nm. In addition, the estimated particle size from the TEM image of the silver nanoparticles after irradiation also increased from 5 to 10 nm when the corresponding sliver concentration increased from 1 to 10 mM. Many researchers have reported that the antimicrobial activity of nano-silver increases with decreasing particle size . Therefore, to synthesize AgNP with desired size for different applications, the selection of the initial Ag + concentration to prepare AgNP is essential.
Figure 1 UV-Vis spectrum (left) and TEM image (center) and size distribution (right) of AgNPs / chitosan with Ag + concentration of 1 (a), 2.5 (b), 5 ( c) and 10 mM (NS).
On the other hand, the effect of Mw of chitosan on the particle size of nano-silver was also studied. The results in Figure 2 show that the Mw of chitosan affects the size of the silver nanoparticles formed in the irradiated samples and the reduction of Mw of chitosan from 550 to 100 kDa leads to an increase in the size of AgNPs from 10 to 15 nm. Although the mechanism of these effects is not fully understood, the increase in the viscosity of the solution corresponding to an increase in Mw chitosan may have reduced the dynamism of AgNP and prevented them from agglomerate to form. larger nanoparticles.
Figure 2 TEM image and size distribution of AgNP stabilized by chitosan with Mw of 100 (a), 250 (b) and 550 kDa (c).
2. In vitro antifungal activity of AgNPs / Chitosan against C. cassiicola
To test the antifungal effect by particle size of AgNPs / chitosan sample, AgNPs with particle size of about 5, 10 and 15 nm were used. As can be seen in Figures 3 and 4, the small particle size has a stronger effect on the growth of C. cassiicola on rubber leaf extract after 192 hours of incubation compared to the large medium. In particular, the addition of 50 ppm nano silver with a particle size of 15 nm showed a strong inhibitory effect on C. cassiicol with a fungal colony diameter of only 43.1 mm compared to 90 mm of the untreated control and The inhibitory effect was calculated to be about 52.1%. The antifungal effect increased to 70.6% when the particle size decreased to 10 nm and reached nearly 100% for the 5 nm particle size. Therefore, the antifungal effect against C. cassiicola increased as the nano silver size decreased. Similar trends have been reported previously by Carlson et al. . In addition, Franci et al.  also showed that smaller AgNPs penetrate the cell wall more easily and alter and inhibit cell signaling pathways as well as DNA replication and damage. cellular organs through oxidative reactions.
Figure 3 Antifungal activity against C. cassiicola of AgNPs with different particle sizes. (a) Diameter of fungal colonies on rubber leaf extract medium after 192 hours of incubation and (b) inhibitory effect.
Figure 4 Growth of C. cassiicola after 192 hours of incubation on rubber leaf extract medium supplemented with AgNPs with different particle sizes.
On the other hand, the antifungal activities of AgNPs have been shown to increase with increasing silver levels [9, 19–21]. In this study, 10 nm-sized AgNPs stable in 1% chitosan (Mw ~ 550 kDa) were used to evaluate in vitro antifungal activities against C. cassiicola at different AgNPs concentrations. Results from Figures 5 and 6 indicate that, after 10 days of incubation, growth inhibition for C. cassiicola on culture plates supplemented with AgNPs 10 and 30 ppm with corresponding inhibitory effects from 6.3 to 18.5% is quite low. Meanwhile, the development of C. cassiicola was clearly inhibited when adding 50 ppm with a fungal colony diameter of only 50 mm compared with 90 mm on the untreated control plate (inhibitory effect ~ 66.4%). However, the inhibitory activity of nano silver increased when the additive concentration increased to 70 ppm (inhibitory efficiency ~ 91.7%) and the growth of C. cassiicola was completely inhibited when the silver concentration reached. 90 ppm with ~ 100% inhibition efficiency.
Figure 5 Antifungal activity against C. cassiicola of AgNPs (10 nm) at different concentrations. (a) Diameter of fungal colonies on rubber leaf extract medium after 192 hours of incubation and (b) inhibitory effect
Figure 6 Growth of C. cassiicola after 216 hours of incubation on rubber leaf extract medium supplemented with different concentrations of AgNP.
3. In Vivo Antifungal activity of AgNPs / Chitosan against C. cassiicola
There are several reports of AgNPs’ antimicrobial effects against bacteria and fungi [9, 22, 23]. However, to date, there have been rare reports of in vivo literature on the effects of AgNPs against fungi in plants. In this study, the antifungal effects of AgNPs on C. cassiicola were directly investigated on the rubber tree. AgNPs with concentrations of 2.5 to 12.5 ppm were applied on the tested rubber trees through foliar fertilization. Additionally, AgNP has been shown to be effective in controlling the disease when sprayed after a fungal infection. The results from Table 1 show the damage of rubber trees infected by C. cassiicola can be minimized with AgNP. In particular, plant DI decreased from 30 to 6.3% by treating AgNPs with concentrations ranging from 2.5 to 12.5 ppm, respectively. Furthermore, it can be seen from Table 1 and Figure 7 that AgNPs treatment also significantly reduced the DI residue of infected plants and that the DI ratio was inversely proportional to the AgNPs treated. These results are consistent with the previous discovery of the fungus Colletotrichum causing anthracnose to pepper reported by Lamsal et al. [ 8 ]. The reason can be explained by the fact that, after spraying, AgNPs can attach to and kill diseased mycelium in plant tissue [24, 25].
Table 1 Effect of AgNPs concentration on the removal of C. cassiicola after being inoculated into rubber trees.
Figure 7 Rubber trees infected with C. cassiicola and treated with AgNPs (10 nm) at different concentrations. Check: plants not treated with fungal spores and AgNPs; Prevention: plants infected with fungal spores before treatment with only distilled water.
The inhibition of AgNPs on C. cassiicola mycelium germination was analyzed via SEM to elucidate the effect of AgNPs on fungal growth. The SEM image in Figure 8 shows that AgNPs have visibly damaged mycelium, while the water-treated mycelium (control) appears to remain intact. Mycelium is sunken and damaged by the action of AgNPs. The mycelium observed on day four after the 7.5–12.5 ppm AgNPs treatment showed deformation in the mycelium growth and the shape of the mycelium walls. The mycelium septum also tore off on the fourth day damaged mycelium and many of the mycelium collapsed.
Figure 8 SEM image of C. cassiicola mycelium treated with AgNPs. The mycelium grows on a rubber leaf extract agar medium plate sprayed with water as a control or with equal volumes of 7.5 and 12.5 ppm of 10 nm AgNPs solution. Photos were taken on the fourth day after the treatment.
Conclusion effective nano silver
AgNPs with different particle sizes have been successfully synthesized by the Co-60 gamma irradiation method using chitosans with different Mw as stabilizers. The synthetic AgNPs / chitosan products showed strong inhibitory effects on C. cassiicola on rubber leaf extract with an antifungal effect of ~ 100% when treated with 50 ppm AgNPs with dimensions of 5 nm or 90. ppm AgNPs with the size of 10 nm. The treatment of leaves with AgNPs 5-12.5 ppm solution strongly reduces the disease rate of rubber trees as well as the disease rate on rubber leaves infected with C. cassiicola fungus. Therefore, AgNPs / chitosan synthesized by gamma Co-60 irradiation is considered a green method that can be used as a potential and effective fungicide for C control. cassiicola, a fungus that causes dangerous diseases on the rubber tree.
Reference source:Radiation Synthesis of Silver Nanoparticles/Chitosan for Controlling Leaf Fall Disease on Rubber Trees Causing by Corynespora cassiicola Le Thi An Nhien,1,2Nguyen Duc Luong,2Le Thi Thuy Tien,2 and Le Quang Luan
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